Jananji, S., Risi, C., Lindamulage, I. K., Picard, L. P., Van Sciver, R., Laflamme, G., Albaghjati, A., Hickson, G. R., Kwok, B. H. and Galkin, V. E. (2017). Multimodal and polymorphic interactions between Anillin and Actin: Their implications for cytokinesis. J Mol Biol [Epub ahead of print]. PubMed ID: 28147230Summary:Cytokinesis of animal cells requires the assembly of a contractile ring, which promotes daughter cell splitting. Anillin is a conserved scaffold protein involved in organizing the structural components of the contractile ring including filamentous actin (F-actin), myosin, and septins and in forming the subsequent midbody ring. Like other metazoan homologs, Drosophila anillin contains a conserved domain that can bind and bundle F-actin, but the importance and molecular details of its interaction with F-actin remain unclear. This study shows that in a depletion-and-rescue assay in Drosophila S2 cells, anillin lacking the entire actin-binding domain (ActBD) exhibits defective cortical localization during mitosis and a greatly diminished ability to support cytokinesis. Using in vitro binding assays and electron microscopy on recombinant fragments, it was determined that the anillin ActBD harbors three distinct actin-binding sites (ABS 1-3). Each ABS binds to a distinct place on F-actin. Importantly, ABS1 and ABS3 partially overlap on the surface of actin and, therefore, interact with F-actin in a mutually exclusive fashion. Although ABS2 and ABS3 are sufficient for bundling, ABS1 contributes to the overall F-actin bundling activity of anillin and enables anillin to switch between two actin-bundling morphologies and promote the formation of three-dimensional F-actin bundles. Finally, it was shown that in live S2 cells, ABS2 and ABS3 are each required and together sufficient for the robust cortical localization of the ActBD during cytokinesis. Collectively, these structural, biochemical, and cell biological data suggest that multiple anillin-actin interaction modes promote the faithful progression of cytokinesis.

Kim, H., Johnson, J. M., Lera, R. F., Brahma, S. and Burkard, M. E. (2017). Anillin Phosphorylation Controls Timely Membrane Association and Successful Cytokinesis. PLoS Genet 13(1): e1006511. PubMed ID: 28081137Evolutionary Homolog Study
During cytokinesis, a contractile ring generates the constricting force to divide a cell into two daughters. This ring is composed of filamentous actin and the motor protein myosin, along with additional structural and regulatory proteins, including anillin (see Drosophila Scraps). Anillin is a required scaffold protein that links the actomyosin ring to membrane and its organizer, RhoA (see Drosophila Rho). However, the molecular basis for timely action of anillin at cytokinesis remains obscure. This study finds that phosphorylation regulates efficient recruitment of human anillin to the equatorial membrane. Anillin is highly phosphorylated in mitosis, and is a substrate for mitotic kinases. Function of 46 residues on anillin previously found to be phosphorylated in human cells was surveyed to identify those required for cytokinesis. Among these sites, S635 was identified as a key site mediating cytokinesis. Preventing S635 phosphorylation adjacent to the AH domain disrupts anillin concentration at the equatorial cortex at anaphase, whereas a phosphomimetic mutant, S635D, partially restores this localization. Time-lapse videomicroscopy reveals impaired recruitment of S635A anillin to equatorial membrane and a transient unstable furrow followed by ultimate failure in cytokinesis. A phosphospecific antibody confirms phosphorylation at S635 in late cytokinesis, although it does not detect phosphorylation in early cytokinesis, possibly due to adjacent Y634 phosphorylation. Together, these findings reveal that anillin recruitment to the equatorial cortex at anaphase onset is enhanced by phosphorylation and promotes successful cytokinesis.

Hirashima, T., Tanaka, R., Yamaguchi, M. and Yoshida, H. (2018). The ABD on the nascent polypeptide and PH domain are required for the precise Anillin localization in Drosophila syncytial blastoderm. Sci Rep 8(1): 12910. PubMed ID: 30150713Summary:
Targeting proteins to regions where they are required is essential for proper development of organisms. For achievement of this, subcellular mRNA localization is one of the critical mechanisms. Subcellular mRNA localization is an evolutionarily conserved phenomenon from E. coli to human and contributes to limiting the regions at which its products function and efficiently supplies substrates for protein translation. During early Drosophila embryogenesis, while 71% of the 3370 mRNAs analyzed have shown prominent subcellular localization, the underlying molecular mechanisms have not been elucidated.
This study revealed that anillin mRNA, one of the localized mRNAs in early Drosophila embryo, localizes to the tip of the pseudo-cleavage furrow in the Drosophila syncytial blastoderm using in situ hybridization combined with immunohistochemistry. Localization analyses with transgenic fly lines carrying a series of deletion mRNAs indicate that this localization is dependent on its own nascent polypeptides including the actin binding domain (ABD). In addition to the mRNA localization, it is revealed that the pleckstrin homology (PH) domain of Anillin protein is also required for its proper localization. Thus, this study indicates that the precise localization of Anillin protein is tightly regulated by the ABD on the nascent polypeptide and PH domain in the Drosophila syncytial blastoderm.

Cytokinesis in animal cells occurs by ingression of a cleavage furrow,
driven by constriction of an actomyosin-based contractile ring coupled to
insertion of new plasma membrane. An important unanswered question is how are
furrow ingression and new membrane deposition coordinated? The
Drosophila embryo provides an interesting model system with which to
address this question and the general cell biology of furrowing. During the
syncytial blastoderm stage, typical cytokinesis does not occur, but transient
furrows ingress to keep mitotic spindles separate. At this stage, pole cells
separate from the yolk mass by a type of cytokinesis that has been little
studied. Later, during a process called cellularization, interconnected
furrows ingress between each nucleus and synchronously partition the embryo
into ~6000 individual cells. After cellularization, conventional cytokinesis starts
in the mitotic domains of the gastrulating embryo
(Field, 2005 and references therein).

Cellularization is more complex than conventional cytokinesis. It requires
an estimated 25-fold increase in surface area during a single cell cycle,
and is thus useful for exploring the coupling between actomyosin contraction
and insertion of new plasma membrane. It also requires ingression in two
different planes. Initially, furrows ingress perpendicularly to the embryo
surface. The tips of cellularization furrows are called furrow canals, and
they interconnect as an almost hexagonal network surrounding each nucleus.
Ingression of the plasma membrane occurs in at least two stages that differ in
rate (initially slow, then fast) and mechanism, with certain mutations
selectively affecting one stage. Later, once the furrow canals pass the
nuclei, ingression also occurs parallel to the embryo surface. The furrow
canals broaden and become almost triangular-shaped in cross-section, and the
network transforms into an almost hexagonal array of contractile rings. These
constrict around the base of each nucleus, individualizing the cells.
Constriction is incomplete. The newly formed cells remain connected to the
yolk mass by a thin neck of cytoplasm or 'stalk' as the embryo initiates
gastrulation movements (Field, 2005 and references therein).

Given their rich contractile biology, Drosophila embryos have been
useful for investigating the mechanism of furrowing. As expected, embryonic
furrows contain the contractile proteins F-actin
and cytoplasmic Myosin II. They also contain septins and Anillin, conserved furrow
components whose function is less clear. Septins were discovered as CDC
mutants in budding yeast and were implicated in animal cytokinesis by analysis
of mutations in the Drosophila septin Peanut. Peanut was later shown to be involved in cellularization. Biochemical investigation in Drosophila embryos showed that septins bind GTP and assemble into heteromeric complexes and filaments. Their
molecular function is unknown, although septins have been implicated in
vesicle trafficking (Field, 2005).

Anillin was originally isolated from Drosophila embryos by
affinity chromatography on F-actin (Field, 1995) and homologs were later found in vertebrates (Oegema, 2000; Straight, 2005) and C. elegans (see Maddox,
2005). Anillin is required for cytokinesis in Drosophila and vertebrate tissue culture cells (Oegema, 2000; Somma, 2002; Kiger, 2003; Echard, 2004; Straight, 2005), but
its function during furrowing remains unclear. Mid1 and Mid2, two proteins with more limited homology to Anillin, play central roles in cytokinesis in S. pombe (Berlin, 2003; Paoletti, 2000; Tasto, 2003; Field, 2005 and references therein).

Anillin is a multi-domain protein that physically interacts with several
other cleavage furrow components in vitro. Its N terminus contains a region
that binds and bundles F-actin (Field, 1995; Oegema,
2000), and a second region that binds phosphorylated cytoplasmic
Myosin II (Straight, 2005). Its C-terminus comprises a predicted PH domain, an ~100
amino acid module often implicated in binding to membranes via inositol lipids
(Lemmon, 2004; Lemmon, 2002). The C-terminal region of vertebrate Anillin was implicated in septin binding by expression of truncated protein and biochemical assays
(Oegema, 2000; Kinoshita, 2002), but the physiological relevance of that proposed interaction was not tested. To explore the function of Anillin, in particular its potential role in coupling cytoskeletal and membrane dynamics, the effects were analyzed of a series of mutations in Anillin on the diverse, cell cycle regulated furrows in the early Drosophila embryo (Field, 2005).

To find mutations in Anillin, P-element insertions were sought near its map
location, 43DE (Field, 1995). The genomic region around the insertion site
in line P3427, was sequenced, revealing a P-element insertion 53 bp upstream of a potential anillin start codon. The scraps gene also maps in this
region. A screen for recessive female sterile mutations
(Schupbach, 1989) isolated six maternal effect alleles in this gene,
designated scrapsRS, scrapsPQ,
scrapsHP, scrapsPE,
scrapsRV and scrapsPB. Females
homozygous for any of these alleles, or trans-heterozygous for any allelic
combination, lay morphologically normal eggs that fail to hatch. Observation
of mutant embryos using bright-field microscopy showed cellularization
defects. Complementation tests over a deficiency of the region revealed a
zygotic function for scraps (Schupach, 1989) and a
later study identified two zygotic alleles, scraps7 and
scraps8 (Heitzler, 1993). Two additional maternal effect alleles,
scrapsB26-35 and scrapsC82-45, have been identified. In complementation tests, it was found that that the P3427 insertion line is allelic
to scraps (Field, 2005).

To confirm that Anillin is the product of the scraps gene, rescue experiments were performed by injecting anillin cDNA. All the embryos from
scrapsRV/RV mothers that received the transgene were
rescued to hatching, and ~90% of these developed into fertile adults. It is concluded that Anillin is essential for embryonic viability and
is the product of the scraps gene. (Field, 2005).

The strongest molecular conclusion from this work is that Anillin is
required to target and maintain septins in cortical structures. Strong
maternal anillin alleles map to the N terminus of the PH domain, the
region of Anillin previously predicted to interact with septins on the basis
of fragment expression (Oegema, 2000) and in vitro binding assays
(Kinoshita, 2002). These alleles cause severe mis-localization of the septin Peanut and also result in a significant reduction in the amount of Anillin in furrow canals
late in cellularization. Thus, the genetic and biochemical data together
suggest that a physical interaction between the PH domain of Anillin and
septin complexes is required to stably recruit both proteins to ingressing
furrow canals. This interdependence is also supported by analysis of Peanut
mutants. In embryos that lack Peanut, F-actin rings failed to form late in
cellularization, similar to strong anillin alleles. Anillin
localization is normal in slow phase, but the protein is progressively lost
from furrow canals during fast phase, showing that Peanut is necessary for
stable localization of Anillin. A role for Anillin in targeting and
stabilizing septins is also supported by studies in C. elegans, where
the Anillin homolog ANI-1 is required to target the septins to the contractile
ring (Maddox, 2005), and in S. pombe (Berlin, 2003; Tasto,
2003), where the Anillin-related protein Mid2 stabilizes
cortical septins in the medial ring (Field, 2005).

PH domains in many proteins have been implicated in lipid binding, and the
amino acid changes in strong anillin alleles fall in the region
of this domain known to interact with lipids. However,
the PH domain of Anillin lacks the positively charged amino acids that mediate
specific binding to phosphoinositides, and GFP fusions to the C terminus of
human Anillin transiently expressed in mammalian cells did not localize to the
plasma membrane, but instead assembled into small septin-containing foci
(Oegema, 2000). It therefore seems possible that the PH domain of Anillin mediates a
protein-protein interaction with septins and not a protein-lipid interaction.
However, Drosophila Anillin can target to the cellularization front
in the absence of Peanut (Adam, 2000) and the C. elegans ortholog ANI-1 can target to the furrow normally in the absence of the septins
(Maddox, 2005). Many PH domains do not bind inositol lipids with high affinity and require
oligomerization or additional motifs within the same protein to impart
membrane localization (Lemmon, 2002). It is therefore possible that the PH domain of Anillin mediates association with membranes by mechanisms other than septin
binding (Field, 2005).

Morphologically, the most dramatic phenotype of strong anillin
alleles was the appearance of sheets of vesicles (that appear as lines in thin
sections) between nuclei during cellularization, in place of the intact,
apposed plasma membranes deposited behind the cellularization front in
wild-type embryos. The presence of some intact membranes in
mutant embryos fixed early in cellularization
is interpreted as evidence that
lateral plasma membranes are initially deposited in anillin mutant
embryos, but they are unstable and subsequently vesiculate. Other membranes in
the mutant embryos, including pole cell and apical plasma membranes, are
unaffected, arguing that vesiculation is not a fixation artifact, and that the
new lateral plasma membranes have a specific requirement for Anillin and
septins for stability. These membranes are special in at least three ways that
might account for their fragility: they assemble very rapidly, by highly
dynamic exo- and endocytosis; they are probably under tension from the ingressing
furrow canals; and they are closely apposed to each other. Anillin might
regulate vesicle trafficking dynamics; for example, decreasing exocytosis
could lead to a build up of tension and membrane fragmentation. Septins have
been argued to regulate exocytosis in mammalian cells but, in this case,
inactivation of septins leads to increased, rather than decreased, exocytosis.
Alternatively, Anillin might directly regulate physical stability of
membranes. Plasma membranes are physically stabilized in most situations by
attachment to a cortical actin cytoskeleton, and loss of Anillin might
destabilize them by weakening the cortex or its attachment to the membrane.
Destabilization of membranes under tension might lead to the fusion of closely
apposed membranes and the lines of vesicles observed are reminiscent of some
stages of programmed cell fusion, e.g. myoblast fusion.
Anillin itself does not localize to the cortex of the apposed plasma
membranes, but it might function to recruit and leave behind other proteins
required for stability under tension. Septins are normally present and
localized ectopically in anillin mutants; although targeting of
F-actin is less affected, its organization in mutants is unknown. Loss of
septins or F-actin bundling could result in a more fragile, or more weakly
attached, cortical cytoskeleton, causing the membranes to fragment as tension
builds up during cellularization. A role for septins in stabilizing membranes
could be tested by TEM of peanut mutant embryos, which are known to
exhibit defects in nuclear positioning similar to those observed in
anillin mutants (Adam, 2000). Roles for Anillin in regulating membrane trafficking
compared with physical stability might be distinguished by live imaging of
membrane markers to measure exo- and endocytosis, and by imaging thermal
fluctuations of the new plasma membranes to estimate their stiffness, as a
function of anillin genotype (Field, 2005).

An interesting aspect of the vesiculation phenotype is the tendency of
plasma membrane-derived vesicles to remain localized in sheets behind the
cellularization front, rather than diffusing away. It is thought that they may be
adhering to the baskets of microtubules that surround each nucleus, that are
unaffected in anillin mutant embryos. Remarkably, the physical
organization of vesicles is sufficient to allow gastrulation movements, even though cellularization has failed completely in terms of generating cells bounded by plasma membranes (Field, 2005).

Although defects in septin recruitment mirrored the allelic series, septin
recruitment is clearly not the only function of Anillin. Similar
defects are observed in the timing and rate of cellularization front ingression in all
alleles examined, including weak alleles that had no obvious defects in septin
recruitment. Defects were also observed in F-actin and Myosin II
localization/organization that are consistent with previously identified
biochemical interactions (Field, 1995; Straight, 2005). Since the alleles did not alter the N terminus of Anillin, where it interacts with F-actin and Myosin II, these defects probably result from reduced localization of functional Anillin. A role for Anillin in
scaffolding contractile structures has been demonstrated in C.
elegans, where the Anillin homolog ANI-1 is required to organize foci
containing Myosin II that pull on the plasma membrane during polarity
establishment (Maddox, 2005). Focusing of actomyosin contraction by Anillin is also
suggested by excessive membrane blebbing and mislocalization of myosin II seen
during cytokinesis after knocking down Anillin by RNAi
(Echard, 2004; Somma, 2002; Straight, 2005; Field, 2005 and references therein).

A more structural role for Anillin may be important late in cytokinesis and
cellularization. Myosin II, and most F-actin, typically leave furrows before
cytokinesis is complete, and it is important that something prevents the
furrow from opening back up when it is no longer actively contractile. Anillin
and septins may assemble into a structure under the plasma membrane to
stabilize the neck of cytoplasm late in cytokinesis. One allele,
anillinPE/PE, exhibited a severe effect on pole cell
formation because of re-opening of the neck of cytoplasm connecting the pole
cell to the yolk mass. Interestingly, this allele exhibited milder defects in
cellularization, was viable over zygotic mutations and thus was termed 'weak'.
The mutation maps to a region in the middle of Anillin not implicated in any
protein interactions. In other systems, removal of Anillin also leads to
defects late in cytokinesis, with furrows reopening (Echard, 2004; Somma, 2002;
Rogers, 2003; Straight, 2005). Anillin and septins are strongly enriched as rings or short tubes in stable intracellular bridges, including male ring canals during spermatogenesis
(Hime, 1996; Robinson, 1996),
and the stalks that connect cells to the yolk mass after cellularization. They
also remain in mid-bodies after the contractile proteins leave during
conventional cytokinesis (Field, 1995). Cumulatively, these data suggests that ring- or
tube-shaped Anillin-septin assemblies [rings are the preferred assembly state
of mammalian septins (Kinoshita, 2002)] may stabilize intracellular bridges to facilitate the completion of normal cytokinesis, and to allow communication between sister
cells following incomplete cytokinesis (Field, 2005).

Given its interaction with multiple conserved furrow proteins, and its functional involvement in both contractility and membrane stability, further study of Anillin is likely to reveal detailed aspects of how the cytoskeleton and membrane systems work together during cytokinesis, and related furrowing processes in embryos (Field, 2005).

Anillin binds nonmuscle myosin II and regulates the contractile ring

The contractile ring protein anillin interacts directly with nonmuscle myosin II and this interaction is regulated by myosin light chain phosphorylation. Despite their interaction, anillin and myosin II are independently targeted to the contractile ring. Depletion of anillin in Drosophila or human cultured cells results in cytokinesis failure. Human cells depleted for anillin fail to properly regulate contraction by myosin II late in cytokinesis and fail in abscission. A role is proposed for anillin in spatially regulating the contractile activity of myosin II during cytokinesis (Straight, 2005).

The anillin protein is a multifunctional component of the cytoskeleton that is recruited to the furrow early in cytokinesis but functions primarily late in cytokinesis to focus contractility at the furrow. Anillin is known to directly interact with actin and contribute to the organization of the septin complex along actin filaments (Field, 1995; Kinoshita, 2002). This study shows that anillin also directly interacts with nonmuscle myosin II. This interaction with myosin II depends upon phosphorylation of myosin II regulatory light chain by MLCK, suggesting that anillin only associates with active myosin II (Straight, 2005).

Whether anillin functions in cytokinesis to recruit activated myosin II to the cleavage furrow was tested. Anillin depletion data in vivo rule out this simple model because myosin II is able to localize to the division site and promote furrow contraction with normal timing in the absence of anillin. Oegema (2000) observed reduction of the initial rate of furrow contraction after inhibiting anillin by antibody injections, but the current depletion data suggest this may have been due to the presence of antibody in the furrow rather than anillin removal. This study also found that anillin targets to the furrow normally when myosin II is depleted, although in this case contraction is completely inhibited. Those data are consistent with previous pharmacological studies where it was shown that inhibition of kinases that regulate cytokinesis interfere with targeting of myosin II, but not of anillin, to the furrow (Straight, 2003). It will be interesting to test in the future where the pathways that target myosin II and anillin diverge. Both require the continual presence of microtubules (Straight, 2003) and probably also activated Rho (Somma, 2002) to target normally (Straight, 2005).

The primary defect observed in cells that lack anillin is a delocalization of contraction at the end of cytokinesis. Observation of myosin II dynamics in anillin-depleted cells revealed that myosin II is no longer constrained to the contractile ring as it is in control cells and instead is found in the cell cortex concomitant with aberrant cell contraction. This aberrant contraction often results in both mispositioning of the cleavage furrow to yield binucleate cells or to furrow regression and thus binucleation. It was not possible to deplete all of the anillin by RNAi in human cells, thus complete depletion or inhibition of the anillin protein may result in an even more severe cytokinesis phenotype. It is not clear whether the phenotype observed represents extra contraction, for example, due to hyperactivation of myosin II, or relocalization of contraction due to mislocalization of active myosin II to ectopic sites. Distinguishing these hypotheses will require measuring contractile properties of the cortex at different positions. Because anillin is restricted to the contractile ring in unperturbed cells, it is unlikely that anillin outside the furrow inhibits myosin II. Overall, these data point to a model whereby anillin binding to activated myosin II restricts its activity to the furrow until cytokinesis can complete. Anillin is retained in the fully contracted furrow much longer than myosin II, and it is also present in intracellular bridges that are no longer contracting (Field, 1995). An extension of this model proposes that loss of myosin II from the fully contracted furrow is promoted by cell cycle-dependent modification of anillin and/or myosin, such as dephosphorylation of myosin regulatory light chain (Straight, 2005).

Several lines of evidence suggest that anillin controls, or at least coordinates multiple aspects of cytokinesis. Two anillin-related proteins in yeast, Mid1 and Mid2, organize distinct steps during cytokinesis. The Mid1 protein, like anillin, relocalizes from the nucleus to the contractile ring early in cytokinesis (Sohrmann, 1996; Wu, 2003). Mid1 mutants are defective in septum placement and formation (Sohrmann, 1996) and overexpression of Mid1 disrupts cytokinesis (Bahler, 1998; Paoletti, 2000). Several important differences exist between Mid1 and anillin. Mid1 is not essential, does not require actin filaments or microtubules to be maintained at the division site, and does not contract with the actomyosin contractile ring (Paoletti, 2000; Wu, 2003). However, Mid1 is important for the initial organization of myosin II at the contractile ring and can interact with myosin II (Wu, 2003; Motegi, 2004). A second anillin-like protein in fission yeast, Mid2, performs other functions that depend on anillin in metazoan cells. In particular, Mid2 organizes septins in fission yeast and is necessary for proper cell separation (Berlin, 2003; Tasto, 2003), whereas metazoan anillin binds directly to septins (Kinoshita, 2002) and participates in targeting septins to the cortex (Oegema, 2000). Mid2 mutant cells have no defect in myosin II localization or contraction at the end of cytokinesis (Berlin, 2003). Thus, metazoan anillin may encompass the activities of both Mid1 and Mid2. It is speculated that the functions of anillin may be split in fission yeast because of the different mechanical requirements for cytokinesis. In yeast, remodeling of the cell wall may be the primary requirement for cytokinesis, whereas cytokinesis in metazoan animals is dominated by the need to physically constrict the equator of the dividing cell. In budding yeast, the mechanical requirements are different again, because the cell division site is predetermined at a narrow constriction. In that system, myosin II targets very early and no anillin-like proteins have been identified (Straight, 2005).

Anillin is known to be essential for the completion of cytokinesis in vertebrate cells and in Drosophila (Somma, 2002). Anillin's interaction with both the septin complex and with filamentous actin may be required for cell abscission. Myosin II leaves the contractile ring late in cytokinesis, but anillin persists at these contracted furrows, suggesting that anillin's role in the completion of cytokinesis may only be partially explained by its interaction with myosin II. The current results suggest an early role for anillin in cytokinesis to properly organize the contractile ring and a late function for anillin in restricting myosin II contraction to the furrow. In Drosophila embryos expressing mutant anillin, actin, and myosin II are disorganized during cellularization. This may reflect an analogous role for anillin in organizing myosin II at the cellularization front as well as at the contractile ring during cytokinesis (Straight, 2005).

The events of mitosis are temporally coupled by the activities of protein kinases that drive the cell cycle and the proteasome that inactivates these kinases and degrades other proteins involved in mitosis. A role has been demonstrated for proteolysis in the disassembly of the contractile ring (Straight, 2003) in vertebrate cells. Possible substrates for this proteolysis are anillin and the cell cycle kinase Polo. In yeast, Mid2 is degraded by ubiquitin-mediated proteolysis (Tasto, 2003), it will be interesting to determine whether in somatic cells anillin is degraded upon mitotic exit, although no change was observed in anillin levels during the metaphase-to-interphase transition in Xenopus egg extracts. Mid1 is controlled by the activity of Polo kinase in fission yeast. In Xenopus extracts, anillin is rapidly dephosphorylated as cells exit mitosis and is efficiently phosphorylated by Polo kinase in vitro. Regulation of anillin by phosphorylation may provide another effective means of coupling the early and late events of cytokinesis to the cell cycle (Straight, 2005).

The results demonstrate a role for anillin in localizing the contractile activity of myosin in addition to anillin's previously identified functions in binding actin and organizing the septins. Thus, anillin seems to be a central factor for coupling the filament systems that interact during cytokinesis. Understanding how proteins such as anillin dynamically organize the cytoskeletal and regulatory networks that are integrated to accomplish cytokinesis will be key to understanding the process of cell division (Straight, 2005).

Cell division requires a direct link between microtubule-bound RacGAP and Anillin in the contractile ring

The mitotic microtubule array plays two primary roles in cell division. It acts as a scaffold for the congression and separation of chromosomes, and it specifies and maintains the contractile-ring position. The current model for initiation of Drosophila and mammalian cytokinesis postulates that equatorial localization of a RhoGEF (Pbl/Ect2) by a microtubule-associated motor protein complex creates a band of activated RhoA, which subsequently recruits contractile-ring components such as actin, myosin, and Anillin. Equatorial microtubules are essential for continued constriction, but how they interact with the contractile apparatus is unknown. This study reports the first direct molecular link between the microtubule spindle and the actomyosin contractile ring. The spindle-associated component, RacGAP50C, which specifies the site of cleavage, interacts directly with Anillin, an actin and myosin binding protein found in the contractile ring. Both proteins depend on this interaction for their localization. In the absence of Anillin, the spindle-associated RacGAP loses its association with the equatorial cortex, and cytokinesis fails. These results account for the long-observed dependence of cytokinesis on the continual presence of microtubules at the cortex (Gregory, 2008).

Previous work has shown that initiation of contractile-ring formation is dependent on a complex between RacGAP, the Rho activator Pebble, and the plus-end-directed microtubule protein Pav-KLP, which accumulates at the equatorial cortex during anaphase. This study reports another crucial interaction involving RacGAP, this time relating to the stability of the contractile ring once its position has been set by equatorial microtubules. It was expected that there must be some link between the microtubule-bound positioning complex and the cortical ring, made of cytoskeletal polymers like actin, myosin, and septins. Previous models proposed that this link is through activated Rho: equatorial RacGAP bringing Pebble to activate Rho, which then recruits the actomyosin contractile apparatus via Diaphanous and Rho-dependent kinases. However, this model does not explain why a newly formed ring remains where it began unless there is something structurally connecting the nascent ring to the spindle. In fact, the relationship between the position of microtubules and the ring is extremely robust, as demonstrated by classic manipulation experiments. Furthermore, inhibitor studies have shown that an ongoing interaction between microtubules and the cortex is critical for cytokinesis to proceed. This study presents the first structural connection between the actin-based ring and the microtubule based positioning mechanism that can explain their relationship. RacGAP, localized to the equatorial microtubules by Pav-KLP, induces contractile-ring formation via Pebble activation of Rho and then anchors the forming contractile ring by binding Anillin, which binds both myosin and actin. It is anticipated that this link ensures not only the stable localization of the ring but also the continued delivery of Rho-activating signals by the RacGAP-linked RhoGEF Pebble (Gregory, 2008).

In the absence of Anillin, phenotypes such as ring slippage have been observed (Straight, 2005) that are strikingly similar to those seen when microtubules are removed, consistent with the model for its role in attaching the ring to cortical microtubules, in order that the cleavage position is dictated by the spindle. The loss of Anillin phenotype described, in which RacGAP is found only on interpolar microtubules, can also be seen in human cells (Zhao, 2005). RacGAP and Pav-KLP are normally found on equatorial microtubules, and this localization is critical for cytokinesis, at least in Drosophila. Evidence from vertebrate cells indicates that contractile-ring formation is similarly dependant on cortical RacGAP (Yuce, 2005). Recent evidence for the role for cytokinesis failure in the generation of aneuploid cells and the promotion of tumorigenesis and the strong conservation of the Anillin sequence across phyla suggests that this newly discovered link between centralspindlin and the contractile ring is likely to be broadly significant (Gregory, 2008).

Interaction between Anillin and RacGAP50C connects the actomyosin contractile ring with spindle microtubules at the cell division site

Anillin, one of the first factors recruited to the cleavage site during cytokinesis, interacts with actin, myosin II and septins, and is essential for proper organization of the actomyosin contractile ring. Affinity-purification methodology coupled with mass spectrometry was used to identify Anillin-interacting molecules in Drosophila cells. Several actin and myosin proteins, three of the five Drosophila septins and RacGAP50C (Tum), a component of the centralspindlin complex, were isolated. Using drug and RNA interference (RNAi) treatments it was established that F-actin is essential for Anillin cortical localization in prometaphase but not for its accumulation at the cleavage furrow after anaphase onset. Moreover, septins are not recruited to the cleavage site in cells in which Anillin os knocked down by RNAi, but localizes to central-spindle microtubules, suggesting that septins travel along microtubules to interact with Anillin at the furrow. Finally, it was demonstrated that RacGAP50C is necessary for Anillin accumulation at the furrow and that the two proteins colocalize in vivo and interact in vitro. Thus, in addition to its role in activating RhoA signalling, RacGAP50C also controls the proper assembly of the actomyosin ring by interacting with Anillin at the cleavage furrow (D'Avino, 2008).

It is surmised that Anillin initially localizes to the cortex after mitotic entry because of its strong affinity for F-actin. Then, after anaphase onset, at least two distinct mechanisms co-operate to localize Anillin at the furrow. The first, mediated by Rho but independent of RacGAP50C, excludes Anillin from the polar regions, probably as a result of cell elongation, as already shown for myosin. The second involves a direct interaction with RacGAP50C, which restricts and maintains the localization of Anillin to the cleavage site during furrow ingression (D'Avino, 2008).

Three of the five Drosophila septins -- Pnut, Sep2 and Septin 5 (Sep5) -- were identified in pull-down assays. A fourth septin, Septin 1 (Sep1), was detected only in one of the two separate purifications and with a very low score, making its identification more uncertain. Although the interaction between Anillin and septins has already been described in another system (Kinoshita, 2002), it was previously unclear which of the five Drosophila septins could complex with Anillin. Interestingly, the two showing the highest scores, Pnut and Sep2, are also the only two septins that were identified in a genome-wide screen for genes required for cytokinesis in Drosophila cultured cells (Echard, 2004). The localization of Pnut and Sep2 were analyzed after Anillin RNAi. Sep5 localization could not be analyzed because no antibodies are currently available. Moreover, its high homology with Sep2 (~70%), and the low score and number of peptides obtained from mass-spectrometry analysis, made its identification as an interacting protein dubious. Western blot analysis indicated that Anillin could not be detected after a 48-hour incubation with dsRNA. Both Pnut and Sep2 failed to localize to the cleavage site in these Anillin-depleted cells, and instead localized to the central-spindle microtubules in a significant percentage (30%-40%) of telophase cells. Because these two septins, unlike Anillin, did not accumulate to the cortex after nuclear envelope break-down, the results suggest that Pnut and Sep2 travel along the central-spindle microtubules to be delivered to the cortex, in which they then interact with Anillin. A similar failure to recruit Pnut to the cleavage furrow was also observed in scraps (Anillin) mutant embryos, although mislocalization to the central spindle was not reported in that study (Field, 2005). Conversely, Anillin localization during cytokinesis was unaffected in cells incubated for 96 hours with dsRNAs directed against either pnut or Sep2, or against both septins simultaneously. Western blot analysis confirmed that the expression of both septins was severely reduced after RNAi treatment. Interestingly, the expression of Pnut was reduced after Sep2 RNAi and vice-versa, suggesting that depletion of one septin can affect the stability of the other, consistent with the observation that these two septins are found in a three-protein complex along with Sep1 (Field, 1996). These results indicated that severe depletion of Pnut and Sep2 did not compromise Anillin localization during cytokinesis. No significant increase in the number of multinucleate cells, however, was detected in septin-depleted cells and telophase figures appeared normal. This is in accord with two previous reports showing that septin RNAi treatments for either 3 or 4 days did not cause cytokinesis failure (Eggert, 2004; Somma, 2002). However, Echard (2004) reported an increase in cytokinesis defects when cells were exposed to dsRNAs against pnut or Sep2 for a longer period (6 days). The difference in incubation time might very well explain the discrepancy between these two conflicting sets of results (D'Avino, 2008).

In summary, it is concluded that, after anaphase onset, Anillin directly binds RacGAP50C to establish a connection between the actomyosin filaments responsible for furrow ingression and a sub-population of spindle microtubule that contact the equatorial cortex, the peripheral microtubules. This interaction appears necessary to restrict and/or maintain the localization of Anillin to the cleavage site during furrowing. In turn, Anillin recruits at least two septins, Pnut and Sep2. These proteins then form a scaffold necessary for proper organization of the actomyosin filaments and their interaction with the plasma membrane (Eggert, 2006). These data also provide a molecular mechanism for the previous observation that a membrane-tethered version of RacGAP50C could generate ectopic furrows that contained both Anillin and Pnut (D'Avino, 2006). Thus, RacGAP50C not only activates RhoA during cytokinesis through its interaction with the RhoGEF Pbl, but also binds Anillin at the furrow to control the proper organization of the actomyosin contractile ring. These results further support a model in which the RacGAP component of the centralspindlin complex acts as a key regulator of furrow formation and ingression, and so represents a major furrow-inducing signal (D'Avino, 2005; Saint, 2003; D'Avino, 2008 and references therein).

Cindr interacts with anillin to control cytokinesis in Drosophila

Cytokinesis, the final step of cell division, conventionally proceeds to cell separation by abscission, or complete cytokinesis, but may in certain tissues be incomplete, yielding daughter cells that are interconnected in syncytia by stable intercellular bridges. The mechanisms that determine complete versus incomplete cytokinesis are not known. This study reports a novel in vivo role of the Drosophila CD2AP/CIN85 ortholog Cindr in both complete and incomplete cytokinesis. Evidence is shown for the presence of persistent intercellular bridges in the major larval imaginal disc epithelia. During conventional division of both cultured and embryonic cells, Cindr localizes to cleavage furrows, intercellular bridges, and midbodies. Moreover, in cells undergoing incomplete cytokinesis in the female germline and the somatic ovarian follicle cell and larval imaginal disc epithelia, Cindr localizes to arrested cleavage furrows and stable intercellular bridges, respectively. In these structures, Cindr colocalizes with the essential cytokinesis regulator Anillin. Cindr interacts with Anillin, and depletion of either Cindr or Anillin gives rise to binucleate cells and fewer intercellular bridges in vivo. It is proposed that Cindr and Anillin cooperate to promote intercellular bridge stability during incomplete cytokinesis in Drosophila (Haglund, 2010).

Because the in vivo functions of the evolutionarily conserved CD2AP (CD2-associated protein) and SH3KBP1 (SH3-domain kinase binding protein 1)/CIN85 (Cbl-interacting protein of 85 kDa) family of multiadaptor proteins remain incompletely understood, this study investigated the sole Drosophila CD2AP/CIN85 ortholog, Cindr (CG31012). Cindr expression and subcellular localization were systematically analyzed at various stages of Drosophila development and in cultured Drosophila S2 cells by using a Cindr antibody. One or both of the two largest Cindr isoforms were detected during oogenesis, embryogenesis, larval development, in adult flies, and in S2 cells by western blot and immunocytochemistry (Haglund, 2010).

In cultured Drosophila cells undergoing cytokinesis, Cindr localized to cleavage furrows, intercellular bridges, and the midbody. After abscission, Cindr could further be detected in midbody remnants. The essential cytokinesis regulator Anillin has previously been shown to localize in a similar manner during cell division, so its putative colocalization with Cindr was analyzed, and indeed the two proteins colocalize throughout cytokinesis. Cindr also colocalizes with actin at the inner rim of the intercellular bridge. To evaluate the functional importance of Cindr during cytokinesis, the cellular phenotypes were studied after Cindr depletion or overexpression. Effective reduction of cindr by RNA interference-mediated gene silencing (RNAi) resulted in an about 3-fold increase in the number of binucleate S2 cells, as compared to cells treated with control dsRNA. During live imaging experiments, it was observed that Cindr-depleted S2 cells displayed a delay in abscission and cleavage furrow regression. Consistently, overexpression of Cindr in human cells caused a dominant-negative effect, with a clear increase in the fraction of binucleate cells and cells in late cytokinesis (Haglund, 2010).

Given the high expression of Cindr in Drosophila embryos, whether Cindr may be involved in conventional cell division during embryogenesis was investigated. In Drosophila embryonic mitotic domains, GFP-tagged endogenous Cindr was found to colocalize with Anillin at contractile rings during early cytokinesis, as well as at midbodies during late cytokinesis. Taken together, these data implicate a role for Cindr in cytokinesis during conventional cell divisions in cultured Drosophila cells and in the embryo (Haglund, 2010).

During oogenesis, Cindr was found expressed in both germ cells and the surrounding somatic follicle cell epithelium. In germ cells, Cindr localizes strongly to small ring-shaped structures in the anterior part of the germarium, which is organized as follows. In region 1, germ stem cell daughter cells, called cystoblasts, undergo four mitotic divisions by incomplete cytokinesis, leading to the formation of a cluster of 16 interconnected germ cells. The 16-cell cluster moves into region 2a, where the arrested cleavage furrows begin maturing into intercellular bridges called ring canals. In order to determine the identity of the Cindr-postive rings, costainings were performed with known cleavage furrow and ring canal components. It was found that Cindr colocalizes with Anillin (CG2092) and Pav-Klp (CG1258), two constituents of arrested cleavage furrows, in region 1 of the germarium. In this area, Cindr localizes strongly to cleavage furrows surrounding fusomes, organelles that branch out in the germ cell cysts during the mitotic divisions. Interestingly, in region 2a, upon fusome breakdown and recruitment of the ring canal markers hts-RC (CG9325) and phospho-tyrosine (pTyr), Cindr disappeared from cleavage furrows. After this point Cindr could not be detected at growing ring canals during the rest of oogenesis, which is interesting, because other known components remain for longer time (e.g., Anillin until stage 3; Pav-Klp, pTyr, and hts-RC throughout oogenesis). It is therefore concluded that Cindr marks arrested cleavage furrows in mitotically active and newly formed germ cell clusters, but disappears from growing ring canals, suggesting a role in cleavage furrow formation and/or arrest, but not in ring canal growth (Haglund, 2010).

In Drosophila, each individual egg chamber is formed through encapsulation of the germ cells by an epithelium of somatic follicle cells, starting in region 2a of the germarium. Interestingly, whereas Cindr disappears from arrested cleavage furrows in the female germline, it abruptly appears in dot-like structures in the forming egg chamber follicle cell epithelium in region 2a and remains in such structures throughout oogenesis. These are present throughout the follicle cell epithelium and consistently localize apically or at the level of the nuclei and at the borders between adjacent follicle cells. They are localized basally of E-cadherin-containing adherens junctions and do not overlap with markers for early (Rab5), late (Rab7), or recycling (Rab11 or Rab4) endosomes. In light of the implication for Cindr in cytokinesis, reports were found that describe incomplete cytokinesis in the follicle cell epithelium, giving rise to follicle cell clusters with apically localized stable intercellular bridges. Indeed, Cindr clearly colocalizes with both Anillin and Pav-Klp, two known stable intercellular bridge components, at intercellular bridges throughout oogenesis. Consistently, ultrastructural investigation by immunoelectron microscopy also demonstrated the presence of Cindr at follicle cell intercellular bridges, where it localizes mainly at their inner rim in transverse, longitudinal, and oblique sections (Haglund, 2010).

Having identified Cindr at stable intercellular bridges during oogenesis, it was next asked whether Cindr would be present at stable intercellular bridges during other stages of Drosophila development. For this purpose, larval imaginal disc epithelia, out of which the wing and leg discs have previously been shown to contain stable intercellular bridges, were examined. Indeed, Cindr localizes to distinct structures present at cell borders that colocalize with Anillin throughout wing and leg discs and is also detected in similar structures in eye and antennal discs. These were confirmed to be intercellular bridges by electron microscopy and were ultrastructurally similar to follicle cell intercellular bridges. Cindr localizes mainly to the inner rim also in wing disc intercellular bridges. These data indicate that all larval imaginal discs examined contain persistent intercellular bridges and that Cindr represents a stable intercellular bridge component during Drosophila development (Haglund, 2010).

Additionally, also in the larval brain, it was found that Cindr localizes at structures present at cell borders that colocalize with Anillin and bridges displaying the characteristic intercellular bridge ultrastructure. Indeed, the larval brain has been suggested to contain stable intercellular bridges, but this remains an area of investigation. Interestingly, intercellular bridges of dividing neuroblasts display a more variable morphology. Consistent with the data in, Cindr moreover localizes to contractile rings in the larval imaginal discs and brain (Haglund, 2010).

The role of Cindr at stable intercellular bridges was examined in vivo. RNAi-mediated cindr depletion in somatic follicle cells results in a significant increase in the number of binucleate cells, as compared to control egg chambers, which contain essentially only mononucleate cells. Similarly, RNAi-mediated knockdown of anillin dramatically increases the number of binucleate follicle cells. Interestingly, the increase in binucleate cells is accompanied by a significant decrease in the average number of follicle cell intercellular bridges per nucleus, whereas the number of bridges compared to the number of cells was essentially unchanged. The higher levels of binucleate cells in Anillin- compared to Cindr-depleted epithelia may be accounted for by a faster depletion of Anillin compared to Cindr protein levels. In Anillin-depleted epithelia, binucleate follicle cells were indeed detected in early egg chamber stages arising from defective cell divisions, whereas binucleate Cindr-depleted cells were detected only at late egg chamber stages after the cease of follicle cell divisions in stage 6. These findings indicate a role for Cindr in stabilizing intercellular bridges in the follicle epithelium during oocyte growth (Haglund, 2010).

To address the molecular mechanisms by which Cindr may act at intercellular bridges, it was finally asked whether Cindr and Anillin may interact, given their similar localization pattern throughout Drosophila development and the presence of a Px(P/A)xxR motif (PLARLR, amino acids 145-150) in Anillin. Such motifs interact with the SH3 domains of mammalian CD2AP/CIN85 family members and in fact CD2AP interacts with such a motif in human Anillin. The SH3 domains of Cindr show high sequence homology with the SH3 domains of CD2AP/CIN85, and in pull-down experiments via four GST-tagged parts of Anillin, Cindr indeed associated with the most N-terminal (GST-A1) PLARLR-containing region of Anillin. Importantly, mutation of the consensus arginine to alanine (R150A) in the motif abolished the interaction with Cindr. An interaction was detected between Cindr and the C-terminal (GST-A4) region of Anillin, which is interesting because CD2AP and human Anillin were reported to associate only via an N-terminal Px(P/A)xxR motif. Taken together, these data indicate that Cindr interacts with Drosophila Anillin via two distinct sites: the PLARLR motif via its SH3 domains and an additional site in the C terminus of Anillin (Haglund, 2010).

In summary, this study identified a novel in vivo role of Cindr as a general component of stable intercellular bridges during Drosophila development. Cindr localizes to arrested cleavage furrows in the female germline and to somatic stable intercellular bridges in the follicle cell and larval imaginal disc epithelia. Only a limited number of such components have previously been described, including Anillin, Pav-Klp, and Mucin-D, and interestingly Cindr interacts with Anillin and colocalizes with it at intercellular bridges throughout Drosophila development. Given the increase in binucleate cells and decrease in intercellular bridge numbers upon Cindr and Anillin depletions in follicle cells, it is proposed that Cindr and Anillin interact to promote intercellular bridge stability in tissues that undergo incomplete cytokinesis. One mechanism may be via stabilization of actin at the inner rim of the bridge, as indicated by the fact that Cindr/CD2AP/CIN85 and Anillin are well-established actin regulators and Cindr indeed localizes to the inner rim of stable follicle cell intercellular bridges that is lined with actin filaments. During conventional cell divisions, Cindr and Anillin may play a similar role, as shown by the fact that Cindr colocalizes with actin in the bridge, and Cindr was also found participating in the final abscission step, like CD2AP. It is noted that the CD2AP/CIN85/Cindr family of proteins may function as scaffold proteins during cytokinesis because of their ability to oligomerize and to associate with Anillin, and possibly septins, MgcRacGAP/RacGAP50C, and MKLP1/Pav-Klp. Finally, the presence was shown of persistent intercellular bridges in the major larval imaginal disc epithelia, suggesting that their development may require coordinated intercellular communication within cellular syncytia. A better understanding of complete and incomplete cytokinesis in vivo, to which these data contribute, not only expands the knowledge about these processes during development, but may also provide clues to how accurate cytokinesis regulation is achieved to prevent cell division defects associated with cancer development (Haglund, 2010).

Opposing actions of septins and Sticky on Anillin promote the transition from contractile to midbody ring

During cytokinesis, closure of the actomyosin contractile ring (CR) is coupled to the formation of a midbody ring (MR), through poorly understood mechanisms. Using time-lapse microscopy of Drosophila melanogaster S2 cells, this study shows that the transition from the CR to the MR proceeds via a previously uncharacterized maturation process that requires opposing mechanisms of removal and retention of the scaffold protein Anillin. The septin cytoskeleton acts on the C terminus of Anillin to locally trim away excess membrane from the late CR/nascent MR via internalization, extrusion, and shedding, whereas the citron kinase Sticky acts on the N terminus of Anillin to retain it at the mature MR. Simultaneous depletion of septins and Sticky not only disrupted MR formation but also caused earlier CR oscillations, uncovering redundant mechanisms of CR stability that can partly explain the essential role of Anillin in this process. These findings highlight the relatedness of the CR and MR and suggest that membrane removal is coordinated with CR disassembly (El Amine, 2013).

Cortical compartments partition proteins and membrane at the
cell surface to define regions of specialized function. This study
asks how cortical compartments arise along the plasma
membrane furrows that cellularize the early Drosophila embryo,
and investigates the influence that this compartmentalization has
on furrow ingression. Zygotic gene product
Nullo aids the establishment of discrete cortical compartments,
called furrow canals, which form at the tip of incipient furrows.
Upon nullo loss-of-function, proteins that are normally
restricted to adjacent lateral regions of the furrow, such as
Neurotactin and Discs large, spread into the furrow canals. At
the same time, cortical components that should concentrate in
furrow canals, such as Myosin 2 (Zipper) and Anillin (Scraps),
are missing from some furrows. Depletion of these cortical
components from the furrow canal compartments precipitates
furrow regression. Contrary to previous models, it was found that
furrow compartmentalization does not require cell-cell junctions
that border the furrow canals. Instead, compartmentalization
is disrupted by treatments that reduce levels of cortical F-actin.
Because the earliest uniform phenotype detected in nullo
mutants is reduced levels of F-actin at furrow canals, it is proposed
that Nullo compartmentalizes furrows via its regulation of F-actin,
thus stabilizing furrows and insuring their ingression to
complete cellularization (Sokac, 2008).

The establishment of compartments around the cortical F-actin/
Myosin 2 array appears to be a conserved feature of furrowing
during conventional cytokinesis, common from yeast, to sea
urchin and Xenopus embryos, to
cultured mammalian cells. Previousanalyses in cellularizing Drosophila embryos similarly suggested that the F-actin/Myosin 2 furrow canals are discrete compartments
that form at the tips of incipient furrows and are maintained as the
furrows ingress. According to the
data in this study, compartmentalization of the cellularization
furrow emerges as an essential cellular mechanism to concentrate
cortical components at the furrow canal and so ensure sustained
furrow ingression. Nullo has been identified as a developmental regulator
that aids compartment establishment and maintenance. Nullo
activity serves to partition proteins along the furrow from the
beginning of cellularization, retaining cortical components such as
Myosin 2 at the furrow canal while excluding the lateral proteins
Dlg and Nrt. In this way, Nullo stabilizes furrows such that they
ingress to convert the syncytial embryo into a primary epithelial sheet (Sokac, 2008).

The data show that Nullo is unlikely to compartmentalize the
cellularization furrow via basal junctions or the cortical scaffold
proteins Anillin or Septin, and instead supports a model whereby
Nullo regulates cortical F-actin to establish and then maintain furrow
canal compartments. In nulloX embryos, furrow canal breaks and
regression occur at only a fraction of furrows. This is in contrast
to the global expression profile of Nullo protein. Discontinuous
furrow canal phenotypes have similarly been reported following
alternative perturbations of cortical F-actin in cellularizing embryos.
For example, Cyto-B injection does not halt furrowing, but rather
induces breaks in the Myosin 2 furrow canal network. RhoGEF2 and diaphanous (dia) mutants, which also have reduced F-actin levels at furrow canals, are also missing some furrow canals. Taken together, it is now suggested that reduced F-actin compromises furrow canal compartments at all furrows. In support of this, it was found that furrow canal morphology is altered at all furrows and lateral
PM components spread into every furrow canal in nulloX mutants.
However, the spreading of lateral components is not sufficient to
precipitate furrow regression. Instead, it is the stochastic loss of
cortical components from the furrow canal that destabilizes furrows
to the extreme that they may regress. In the case of nulloX embryos,
it was found that even in the absence of furrow canal components such
as Myosin 2, as long as the furrow canal contains some F-actin,
the associated furrow continues to ingress. But these furrows appear
to be sensitized and perhaps the continued dilution of furrow canal actin by the spreading lateral PM components eventually precipitates their regression (Sokac, 2008).

Several published results now suggest that, in addition to Nullo,
the Rho1 GTPase may contribute to furrow compartmentalization.
Rho1 and its activator, RhoGEF2, localize to furrow canals and the
lateral furrow membrane during cellularization, suggesting that
Rho1 is specifically activated there. RhoGEF2 localization is independent of F-actin, and in RhoGEF2 mutants Dia fails to accumulate at the furrow canal and the embryos have reduced levels of cortical F-actin. Interestingly, RhoGEF2 and dia mutants show
phenotypes strikingly similar to nulloX mutants in that some furrow
canals are missing. This supports the assertion that cortical F-actin helps to
maintain furrow canal integrity. Furthermore, following RNAi
depletion of nullo in RhoGEF2 mutants, F-actin does not assemble
at furrow canals, and so the two proteins may function in separate but parallel pathways. Thus a model is favored whereby the combined activities of Nullo and Rho1 provide the full complement of F-actin at and around the furrow canal, which is in turn required to establish and/or maintain the furrow canal compartment (Sokac, 2008).

Why would reduced cortical F-actin compromise furrow
compartmentalization? One possibility is that F-actin recruits and/or
retains particular cytoskeletal or scaffold proteins at the furrow canal
that are required for compartment establishment and/or maintenance.
In fact, in nulloX and Cyto-D-treated embryos, Myosin 2, Anillin
and Septin are missing from some furrow canal compartments. This
mechanism is consistent with that of the axon initial segment, where
PM compartments develop by the progressive recruitment of the
scaffold protein Ankyrin G that tethers cortical F-actin/Spectrin to
the PM. The resulting meshwork traps and concentrates
additional proteins in the compartment, including transmembrane
receptors and ion channels. Alternatively, F-actin levels might control membrane
trafficking events that occur at the furrow canal compartment. In
support of this, cytoplasmic Myosin 2 punctae are seen following F-actin
perturbation in cellularizing embryos, which
might represent some form of trafficking intermediate. Since
cortical F-actin modulates both endocytosis and exocytosis, changes
in actin level might change the rates of membrane and/or protein
uptake and delivery within PM compartments. Lastly, F-actin levels at the furrow canal
might promote lipid heterogeneities at the PM in the form of lipid
micro-domains or rafts, as has been reported in a spectrum of
mammalian cell types and in sea urchin embryos.
Lipid rafts may then define compartments by virtue of their intrinsic
chemical properties or by recruiting additional signaling or scaffolding complexes. Of course, during cellularization these mechanisms may not be mutually exclusive, but should converge on the formation and maintenance of discrete furrow canal compartments that stabilize furrows and ensure their sustained ingression (Sokac, 2008).

Rho-dependent control of anillin behavior during cytokinesis

Anillin is a conserved protein required for cytokinesis but its molecular function is unclear. Anillin accumulation at the cleavage furrow is Rho guanine nucleotide exchange factor (GEF)Pbl-dependent but may also be mediated by known anillin interactions with F-actin and myosin II, which are under RhoGEFPbl-dependent control themselves. Microscopy of Drosophila S2 cells reveal here that although myosin II and F-actin do contribute, equatorial anillin localization persists in their absence. Using latrunculin A, the inhibitor of F-actin assembly, a separate RhoGEFPbl-dependent pathway was uncovered that, at the normal time of furrowing, allows stable filamentous structures containing anillin, Rho1, and septins to form directly at the equatorial plasma membrane. These structures associate with microtubule (MT) ends and can still form after MT depolymerization, although they are delocalized under such conditions. Thus, a novel RhoGEFPbl-dependent input promotes the simultaneous association of anillin with the plasma membrane, septins, and MTs, independently of F-actin. It is proposed that such interactions occur dynamically and transiently to promote furrow stability (Hickson, 2008).

Drosophila S2 cell lines expressing anillin-GFP were generatated. The anillin-GFP fusion rescued loss of endogenous anillin and its localization paralleled that of endogenous anillin. In interphase it was nuclear, at metaphase it was uniformly cortical, and in anaphase it accumulated at the equator while being lost from the poles.
In some highly expressing cells, nuclear anillin-GFP formed filaments not ordinarily seen with anillin immunofluorescence, but these disassembled upon nuclear envelope breakdown and the overexpression had no appreciable effect on the progress or success of cytokinesis (Hickson, 2008).

Tests were performed to see whether RhoGEFPbl contributes to anillin localization during cytokinesis. After 3 d of RhoGEFPbl RNAi or Rho1 RNAi, anillin-GFP was found to be localized to the cortex in metaphase but does not relocalize to the equator during anaphase, indicating a requirement for RhoGEFPbl, consistent with prior analysis of fixed RhoGEFPbl mutant embryos. Because anillin can bind F-actin (Field, 1995) and phosphorylated myosin regulatory light chain (MRLC; Straight, 2005), RhoGEFPbl might regulate anillin indirectly through its control of F-actin and myosin II (Hickson, 2008).

Latrunculin A (LatA) was used to test whether F-actin was required for anillin-GFP localization. A 30-60-min incubation of 1 µg/ml LatA abolished cortical anillin-GFP localization in metaphase, indicating an F-actin requirement at this phase. However, when anillin normally relocalizes to the equator (~3-4 min after anaphase onset), anillin-GFP formed punctate structures that became progressively more filamentous over the next few minutes, reaching up to several micrometers in length and having a thickness of ~0.3 µm. These linear anillin-containing structures contained barely detectable levels of F-actin and formed specifically at the plasma membrane and preferentially at the equator, although subsequent lateral movement often led to a more random distribution. Thus, anillin responds to spatiotemporal cytokinetic cues even after major disruption of the F-actin cytoskeleton. A substantial (albeit incomplete) reacquisition of cortical phalloidin staining was observed in cells fixed after washing out the drug for a few minutes. In live cells, LatA washout immediately after formation of the anillin structures allowed the preformed structures to migrate from a broad to a compact equatorial zone as the cells attempted to complete cytokinesis. This movement indicates that an F-actin-dependent process can contribute to the equatorial focusing of anillin (Hickson, 2008).

The influence of RhoGEFPbl on anillin behavior was tested in LatA. After RNAi of RhoGEFPbl or Rho1, anillin-GFP remained cytoplasmic through anaphase. Thus RhoGEFPbl and Rho1 are required for anaphase anillin behavior, whether the cortex is intact or disrupted by LatA treatment (Hickson, 2008).

Tests were performed to see whether myosin II impacts anillin-GFP localization. Compared with controls, RNAi of the gene encoding MRLC spaghetti squash (MRLCSqh) inhibited cell elongation during anaphase, slowed furrow formation, and delayed and diminished the equatorial localization of anillin-GFP. However, unlike after RhoGEFPbl RNAi, equatorial accumulation of anillin-GFP was not altogether blocked. It was still recruited but in a broad zone. Furthermore, in the presence of LatA, MRLCSqh RNAi did not affect the formation of the anillin-GFP structures. It is concluded that myosin II contributes to the equatorial focusing of anillin when the F-actin cortex is unperturbed but that myosin II is dispensable for anillin behavior in LatA (Hickson, 2008).

Collectively, these data suggest that multiple RhoGEFPbl-dependent inputs control anillin localization. The slowed equatorial accumulation of anillin when myosin II function was impaired indicates a myosin II-dependent input. That reassembly of the cortical F-actin network (after washout of LatA) allowed preformed anillin structures to move toward the cell equator indicates an F-actin-dependent input. This is consistent with the concerted actions of myosin II and F-actin driving cortical flow, as observed in other cells, and is reminiscent of the coalescence of cortical nodes during contractile ring assembly in Schizosaccharomyces pombe. However, the F-actin- and myosin II-independent behavior of anillin in LatA indicates an additional RhoGEFPbl-dependent input. Thus, RhoGEFPbl can control anillin behavior in anaphase via a previously unrecognized route. Immunofluorescence analysis revealed extensive colocalization between endogenous Rho1 and anillin-GFP in LatA, indicating that Rho1 was itself a component of these structures. These findings are consistent with the idea that Rho1 and anillin directly interact (Hickson, 2008).

Myosin II localization was studied, since it can bind anillin and is controlled by RhoGEFPbl. MRLCSqh-GFP is able to localize to the equatorial membrane independently of F-actin, and in doing so forms filamentous structures resembling those observed with anillin-GFP. Indeed, anillin and MRLCSqh (detected as either MRLCSqh-GFP or with an antibody to serine 21-phosphorylated pMRLCSqh) colocalize (, although they were often offset as if labeling different regions of the same structures (Hickson, 2008).

The effects of anillin RNAi on MRLCSqh-GFP localization were tested. MRLCSqh-GFP recruitment and furrow initiation appeared normal, but within a few minutes of initiation, furrows became laterally unstable and oscillated back and forth across the cell cortex, parallel to the spindle axis, in repeated cycles, each lasting ~1-2 min and eventually subsiding to yield binucleate cells after ~20 min. The phenotype was very similar to that reported for anillin RNAi in HeLa cells (Straight, 2005; Zhao, 2005) and represents a requirement for anillin at an earlier stage than previously noted in Drosophila (Somma, 2002; Echard, 2004). Thus, a conserved function of anillin is to maintain furrow positioning during ingression (Hickson, 2008).

In LatA, anillin RNAi did not prevent equatorial MRLCSqh-GFP recruitment, but instead of appearing as persistent linear structures distorting the cell surface, a more reticular and dynamic structure lacking cell surface protrusions was observed. Thus myosin II can localize independently of both anillin and F-actin but the filamentous appearance of myosin II in the presence of LatA requires anillin, indicating that anillin can influence myosin II behavior in the absence of F-actin, whereas myosin II appeared capable of influencing anillin behavior only in the presence of F-actin (Hickson, 2008).

Septins are multimeric filament-forming proteins that can bind anillin in vitro and function with anillin in vivo (Kinoshita, 2002; Field, 2005; Maddox, 2005; Maddox, 2007). Using an antibody to the septin Peanut, it was found that in nontransfected S2 cells, septinPnut localized to the cleavage furrow and midbody where it colocalized with anillin. Unexpectedly, the septinPnut antibody also strongly labeled bundles of cytoplasmic ordered cylindrical structures, each ~0.6 µm in diameter and of variable length (up to several micrometers). These staining patterns could be greatly reduced by septinPnut RNAi and were thus specific. The cylindrical structures did not appear to be cell cycle regulated, as they were apparent in interphase, mitotic, and postmitotic cells. They also did not colocalize with anillin, nor did their stability rely on anillin. Incubation with 1 µg/ml LatA before fixation inevitably led to disassembly of most of these large structures; however, the resulting distribution of septinPnut depended on the cell cycle phase. In LatA-treated interphase cells, when anillin is nuclear, septinPnut formed cytoplasmic rings, ~0.6 µm in diameter, which are similar to the Septin2 rings seen in interphase mammalian cells treated with F-actin drugs or in the cell body of unperturbed ruffling cells (Kinoshita, 2002; Schmidt, 2004). In LatA-treated mitotic cells, septinPnut was diffusely cytoplasmic (or barely detectable) in early mitosis, whereas in anaphase/telophase, it localized to the same plasma membrane-associated anillin-containing filamentous structures (Hickson, 2008).

Anillin behavior was analyzed after septinPnut RNAi. Although unable to fully deplete septinPnut, it was found that anillin could localize to the equatorial cortex in regions devoid of detectable septinPnut, which is consistent with findings in C. elegans (Maddox, 2005). Importantly, in septinPnut-depleted cells, anillin-GFP still localized to the plasma membrane in LatA but no longer appeared filamentous, indicating that septinPnut is essential for the filamentous nature of the structures and that Rho1 can promote the association of anillin with the plasma membrane independently of septinPnut. However, in this case the plasma membrane to which anillin-GFP localized subsequently exhibited unusual behavior. It was internalized in large vesicular structures, apparently in association with midzone MTs. Although this phenomenon is not understood, it may be related to events induced by point mutations in the septin-interacting region of anillin that give rise to abnormal vesicularized plasma membranes during Drosophila cellularization (Hickson, 2008).

The effects were tested of anillin RNAi on the localization of septinPnut. Using Dia as a furrow marker, 3 d of anillin RNAi prevented the furrow recruitment of septinPnut. In LatA-treated cells, anillin RNAi did not affect the formation of septinPnut rings in interphase cells, but it greatly reduced the formation of septinPnut-containing structures during anaphase/telophase. Thus, anillin is required for the furrow recruitment of septinPnut and for the formation of septinPnut-containing structures in 1 µg/ml LatA. In contrast, Dia could still localize to the equatorial plasma membrane after combined anillin RNAi and LatA treatment, indicating that it can localize independently of both anillin and F-actin. Thus, although Dia partially colocalized with anillin in LatA, this likely reflected independent targeting to the same location rather than an association between anillin and Dia (Hickson, 2008).

These data argue that Rho1, anillin, septins, and the plasma membrane participate independently of F-actin in the formation of a complex. However, anillin, septins and F-actin can also form a different complex in vitro, independently of Rho (Kinoshita, 2002). Perhaps two such complexes dynamically interchange in vivo (Hickson, 2008).

The involvement of MTs in anillin behavior was tested in LatA. Overnight incubation with 25 µM colchicine effectively depolymerized all MTs in mitotic cells and promoted mitotic arrest, as expected. Using Mad2 RNAi to bypass the arrest, anillin-GFP was observed during mitotic exit in the absence of MTs and in the presence of LatA.
Under such conditions, anillin-GFP formed filamentous structures very similar to those formed when MTs were present, indicating that MTs were dispensable for their formation. However, the structures appeared uniformly around the plasma membrane rather than restricted to the equatorial region, which is consistent with the role MTs play in the spatial control of Rho activation (Hickson, 2008).

The LatA-induced anillin structures localize to the ends of nonoverlapping astral MTs directed toward the equator. Live imaging of cells coexpressing cherry-tubulin and anillin-GFP revealed bundles of MTs associating with the filamentous anillin-GFP structures as they formed. Colocalization between anillin-GFP structures and MT ends persisted over many minutes, even after considerable lateral movement at the membrane. Thus, although the anillin structures formed independently of MTs, they stably associated with MTs. These findings support prior biochemical evidence for interactions of MTs with both anillin and septins (Sisson, 2000) and reveal a potential positive-feedback loop in which MTs directed where Rho1-anillin-septin formed linear structures at the plasma membrane, whereas the structures in turn associated with the MT ends. An MT plus end-binding ability of anillin-septin could explain the furrow instability phenotype elicited by anillin RNAi. Accordingly, anillin may physically link Rho1 to MT plus ends during furrow ingression, thereby promoting the focusing and retention of active Rho1, thus stabilizing the furrow at the equator (Hickson, 2008).

These live-cell analyses highlight an unusual behavior of the Rho-dependent anillin-containing structures at the plasma membrane. Initially forming beneath and parallel to the plasma membrane, the structures then often lifted on one side to appear perpendicular to the cell surface while remaining anchored at their base by MTs. This reorientation is interpreted as reflecting avid binding to and subsequent envelopment by the plasma membrane. Although intrinsically stable, the structures exhibited dynamic movement within the plane of the plasma membrane and were capable of sticking to one another, via their ends, giving rise to branched structures that were also capable of breaking apart. Anillin has a pleckstrin homology domain within its septin-interacting region and a membrane-anchoring role of anillin has long been postulated (Field, 1995). These data support such a role and suggest that it is controlled by Rho (Hickson, 2008).

The data highlight the complexity of RhoGEFPbl signaling and lead to a model in which multiple Rho-dependent inputs synergize to control anillin behavior during cytokinesis (Hickson, 2008).
At the appropriate time and location of normal cytokinesis, several proteins, including Rho1, MRLCSqh, Dia, anillin, and SeptinPnut, localized to the equatorial membrane in the presence of LatA. Of these (and apart from Rho1 itself), anillin and SeptinPnut were uniquely and specifically required for the formation of the linear filamentous structures describe in this study. The behaviors of these structures are consistent with prior studies of ordered assemblies of septins and anillin (Oegema, 2000; Kinoshita, 2002) and of interaction between anillin and MTs (Sisson, 2000). Although such structures are not normally seen in furrowing cells, anillin localizes to remarkably similar filamentous structures in the cleavage furrows of HeLa cells (Straight, 2005) arrested with the myosin II inhibitor blebbistatin (Hickson, 2008).

It seems unlikely that LatA induces the described structures through nonspecific aggregation of proteins. Rather, it is proposed that LatA blocks a normally dynamic disassembly of Rho1-anillin-septin complexes (by blocking an F-actin-dependent process required for the event) and that continued assembly promotes formation of the linear structures. Because blebbistatin slows F-actin turnover, blebbistatin and LatA may have induced filamentous anillin-containing structures via a common mechanism. A dynamic assembly/disassembly cycle involving anillin could promote transient associations between the plasma membrane and elements of the contractile ring and MTs, properties that could contribute to furrow stability and plasticity. Finally, because local loss of F-actin accompanies and may indeed trigger midbody formation, LatA treatment may have stabilized events in a manner analogous to midbody biogenesis and could therefore be useful in understanding this enigmatic process (Hickson, 2008).

Cytokinesis entails cell invagination by a contractile actomyosin ring. In epithelia, E-cadherin-mediated adhesion connects the cortices of contacting cells; thus, it is unclear how invagination occurs, how the new junction forms, and how tissue integrity is preserved. Investigations in Drosophila embryos first show that apicobasal cleavage is polarized: invagination is faster from the basal than from the apical side. Ring contraction but not its polarized constriction is controlled by septin filaments and Anillin. Polarized cleavage is due instead to mechanical anchorage of the ring to E-cadherin complexes. Formation of the new junction requires local adhesion disengagement in the cleavage furrow, followed by new E-cadherin complex formation at the new interface. E-cadherin disengagement depends on the tension exerted by the cytokinetic ring and by neighboring cells. This study uncovers intrinsic and extrinsic forces necessary for cytokinesis and presents a framework for understanding how tissue cohesion is preserved during epithelial division (Guillot, 2013).

Epithelial cells divide in the plane of the tissue, allowing the equal partitioning of polarity proteins. This study delineated two major events during epithelial cytokinesis that shed light on how this is controlled. Cleavage progresses along the apicobasal axis and is polarized, as it is faster from basal to apical. This is not due to polarized contraction of the ring but to apical anchoring of the ring to E-cad complexes. Second, cleavage occurs in the plane of junction and involves local adhesion disengagement. In contrast to standard cytokinesis, this study delineated intrinsic and extrinsic mechanical processes operating during epithelial cytokinesis. Contractility of the ring itself is dependent on septins and Anillin. Ring contraction is resisted by intercellular adhesion mediated by E-cadherin complexes and by tension from neighboring cells transmitted by adhesion. Thus, E-cad-based adhesion plays a pivotal role in epithelial cytokinesis by anchoring the contractile ring, while its disengagement uncouples intrinsic and extrinsic tensile activity (Guillot, 2013).

In Drosophila embryos, epithelial cells exhibit polarized cleavage furrow ingression. This is likely to be general in epithelial cells, albeit at different magnitudes. MDCK cells too divide from the basal side toward the apex, and neuroepithelial cells in vertebrates partition the basal body first before the more apical part of the cell. Polarized cleavage is not a property unique to epithelial cells, however. Embryonic cleavage in several species exhibit a range of patterns, from completely unilateral cleavage, as reported in jellyfish (Clytia and Beroe) and Ctenophores (Pleurobrachia), to partly asymmetric cleavage in the one-cell-stage C. elegans embryos). In the latter case, polarized ingression of the cleavage furrow is stochastic and correlates with heterogeneities in the recruitment of the actin crosslinker Anillin and of septins. In anillin and septin knockdowns, cleavage becomes symmetric. This contrasts with activators of MyoII, such as Rho kinase, which affects the speed of contraction but not its polarity. Thus, in nonepithelial cells, polarized cleavage is a purely autonomous process governed by heterogeneities in regulators of contractility. This study found, however, that in Drosophila embryos, polarized cleavage is not determined by polarized distribution of Anillin and septins or by differential biomechanical properties of the ring. Septins display a marginal yet significant enrichment basally, and Anillin is slightly enriched apically. However, invagination was still normally polarized along the apicobasal axis in both peanut mutants and anillin RNAi embryos, despite strong reduction in constriction rat. Moreover, no significant difference between apical and basal relaxation kinetics was detected following ablation in wild-types. The ablation kinetics reflects the relative effect of stiffness in the ring and friction internal to the ring and with the cytoplasm. With the caveat that the latter cannot be directly measured and and is assumed to be uniform, these ablation experiments indicate the relative stiffness in the ring. The fact that relaxation is faster (<5 s) than turnover of the internal components of the ring, such as MyoII, substantiates the idea that mostly the elastic relaxation of the ring was measured and not a quasi-static relaxation associated with turnover/movements of ring components (Guillot, 2013).

The rate of constriction was monotonic such that big rings and small rings contracted at a constant rate in wild-types but also in anillin or septin mutants, although it was strongly reduced in the latter cases. This contrasts with reports in C. elegans, where constriction was scaling with ring size, suggesting a mechanism based on disassembly of contractile units whose number scales with ring size. This difference may stem from the fact that cytokinesis is especially rapid in Drosophila embryos (about 150 s). Alternatively, it could reflect the epithelial nature of the divisions reported in this study (Guillot, 2013).

The evidence argues instead that polarized ingression depends largely on apical anchoring of the ring to E-cad complexes. First, E-cad complexes colocalize with the contractile ring for the most part of invagination. Second, ingression is symmetric in either e-cad or α-cat RNAi embryos. Although E-cad complexes, in particular α-cat, can recruit regulators of MyoII, this cannot explain polarized invagination of the ring, since apical and basal relaxations are not significantly different in wild-types and in α-cat RNAi embryos. E-cad complexes transmit actomyosin tension in epithelia. Two sets of observation support the idea that junctions exert pulling forces on the ring due to anchoring. The ring is stretched laterally as it constricts, and this requires apical junctions via e-cad and α-cat. The relative deformation of the ring following ablation is larger apically than basally, and this also requires cell junctions. It is striking that extrinsic and intrinsic regulators of the ring contraction have very different effects on ring dynamics. In the absence of Pnut or Anillin, the ring constriction is reduced but it is still polarized. However, following e-cad or α-cat depletion, ring constriction is normal but symmetric. It is concluded that the mechanical connection of E-cad complexes to the contractile ring causes polarized invagination. It is possible that, in other systems, both intrinsic and extrinsic regulation will operate in parallel to increase the cleavage asymmetry. This may be important in highly columnar epithelial cells or when adhesion is lower and unable to resist the ring tension (Guillot, 2013).

Polarized cleavage effectively separates apical and basal cleavage, adhesion complexes being a barrier separating the apical and lateral domains. The central problem becomes: How does cleavage occur at adherens junctions? This study delineated two critical phases in junctional cleavage. First, the adherens junctions invaginate with the actomyosin ring, consistent with the fact that the ring is anchored to the junctions. During this phase, E-cad intercellular adhesion is stable in the face of the tension exerted by the ring, and E-cad colocalizes with the ring at the point of coupling. Invagination of junctions then stops as E-cad levels decrease in this area. However, ring constriction continues and appears to detach from junctions. This is interpreted as a point of adhesion disengagement. Adhesion disengagement marks the formation of the new vertices and of the new junction between daughter cells. Electron microscopy images show this membrane disengagement. Consistent with this, the membrane still invaginates with the actomyosin ring), although E-cad is still not detected. Closer examination shows that E-cad monomers are present at this late stage of cytokinesis but that adhesion complexes form gradually from this stage onward. It is striking that adhesion is very locally (<1 μm out of ∼40 μm of junction perimeter) and transiently (∼200 s) perturbed during division. In the first 150 s, E-cad clusters immediately adjacent to the cleavage furrow remain in position as the junction invaginates. This suggests that the cortex can be extensively remodeled locally. It likely reflects the fact that tension induces membrane flows with respect to the actin-rich cortex and argues that E-cad-mediated adhesion does not prevent membrane flow during disengagement. Interestingly, local disengagement allows local cell deformation without affecting the overall shape of cell contacts. Consistent with the idea that adhesion is locally disengaged, the amount of E-cad has a strong impact on the timing and depth of junctional cleavage. Increasing E-cad delays disengagement (i.e., the formation of the new junction, inducing strong cell deformations. More generally, this implies that increasing adhesion may provide an efficient mechanism to prevent local cell-cell disengagement when internal tension is used to remodel junctions during morphogenesis. In apical constriction in the Drosophila mesoderm, actomyosin cables pull on the junctional cortex and reduce junction lengths. If adhesion was not strong enough, local disengagement would occur and junctions could not remodel. The fact that adhesion disengagement is local and transient during cytokinesis is also probably key to the overall maintenance of cell polarity and adhesion during epithelial division (Guillot, 2013).

It is proposed that adhesion disengagement is mechanically induced by tension in the cytokinetic ring and by tension from neighboring cells. When the cumulated tension is higher that the adhesive force, disengagement occurs. Consistent with this, disengagement and formation of the new junction is strongly delayed in mutants that reduce the constriction of the cytokinetic ring, namely, in septin mutants and in Anillin knockdown embryos. Likewise, ablation of neighboring cells delays disengagement. It is, however, possible that adhesion is also locally disrupted by either E-cad endocytosis or phosphorylation of β-cat/Arm (Guillot, 2013).

Adhesion complexes transmit cell tension exerted by neighboring cells. Surrounding junctions and, more specifically, MyoII cables oriented toward or near the cleavage furrow strongly affect furrow invagination when E-cad is present at high levels. The invagination in this case is very shallow, suggesting a tug of war between intrinsic (ring contraction) and extrinsic tension (MyoII cables in neighbors). This results in asymmetric furrows in the plane of junctions due to the asymmetric distribution of MyoII cables around the cell. When E-cad is expressed at lower levels, even if surrounding junctions are oriented toward the cleavage furrow, invagination is unaffected and symmetric. It is proposed that E-cad complexes sensitize cells to their mechanical environment. This may provide a mechanism for cells to integrate stress coming from the environment. It will be important to explore how E-cad levels may affect cells responsiveness to extrinsic stress during division by affecting the timing of the formation of the new junction by local disengagement and the resulting cell shape and topology (Guillot, 2013).

Drak is required for actomyosin organization during Drosophila cellularization

The generation of force by actomyosin contraction is critical for a variety of cellular and developmental processes. Nonmuscle myosin II is the motor that drives actomyosin contraction, and its activity is largely regulated by phosphorylation of myosin regulatory light chain. During the formation of the Drosophila cellular blastoderm, actomyosin contraction drives constriction of microfilament rings, modified cytokinesis rings. This study found that Death-associated protein kinase related (Drak) is necessary for most of the phosphorylation of myosin regulatory light chain during cellularization. Drak was shown to be required for organization of myosin II within the microfilament rings. Proper actomyosin contraction of the microfilament rings during cellularization also requires Drak activity. Constitutive activation of myosin regulatory light chain bypasses the requirement for Drak, suggesting that actomyosin organization and contraction are mediated through Drak's regulation of myosin activity. Drak also is involved in the maintenance of furrow canal structure and lateral plasma membrane integrity during cellularization. Together, these observations suggest that Drak is the primary regulator of actomyosin dynamics during cellularization (Chougule, 2016).

Tight regulation of actomyosin is likely critical for many cellular processes, but how this is accomplished is as yet poorly understood. A key input to the regulation of myosin II is through phosphorylation of the Serine-19, or the Serine-19 and Threonine-18 residues of MRLC (Spaghetti squash). The variety of MRLC kinases might allow different specific aspects of actomyosin dynamics, such as localization, organization and contraction to be regulated independently. Such a system would provide greater flexibility and control than either a single kinase, or multiple kinases acting in concert, regulating all of these functions. drak was found to be required for the organization of myosin II into contractile rings, but is not required for localization of myosin to the cellularization front. Since the majority of Sqh phosphorylation during cellularization is dependent on drak activity, Drak either regulates most aspects of myosin II dynamics during cellularization, or Drak-regulated myosin II organization is required for further function of myosin II, such as contraction (Chougule, 2016).

Myosin II is somewhat less disorganized and Sqh phosphorylation is slightly increased during late cellularization in drak mutants, suggesting that phosphorylation of myosin II by other kinases occurs during late cellularization. Thus other kinases might act synergistically with Drak to regulate actomyosin organization during late cellularization. For example, Drak function has been shown to be partially redundant with Rok function during later development. An alternative possibility is that other kinases that do not normally function in myosin II organization in the microfilament rings might phosphorylate Sqh to some degree and lead to some organization of myosin II in the absence of Drak activity (Chougule, 2016).

Myosin II has been implicated in actin bundling and F-actin organization in some contexts. Since F-actin appears to
be organized normally within drak mutant microfilament rings during early cellularization, it is concluded that myosin II does not play a role in initially organizing F-actin within the microfilament rings during cellularization. F-actin is somewhat disorganized during late cellularization in drakdel mutant embryos, but not as severely as myosin II, nor does the pattern of F-actin distribution fit the pattern of myosin II distribution in drakdel mutant embryos. These observations suggest that F-actin disorganization is an indirect consequence of Drak regulation of myosin II activity, and that F-actin disorganization might be due to actomyosin contraction defects or furrow canal structural defects (Chougule, 2016).

Anillin is required for the organization of actomyosin contractile rings during cellularization and cytokinesis. scraps (scra, anillin) mutant embryos have a myosin II organization defect somewhat similar to that of drak mutant embryos: myosin II is found in discrete bars in the actomyosin network. Despite this similarity, myosin II defects differ between scra and drak mutant embryos. Myosin II becomes more disorganized during late cellularization in scra mutant embryos. Myosin II becomes slightly better organized during late cellularization in drak mutant embryos. This organizational difference is likely caused by actomyosin contraction during microfilament ring constriction occurring in a highly disorganized cytoskeleton in scra mutant embryos, and occurring in a disorganized cytoskeleton that has slightly improved during constriction in drak mutant embryos. Anillin only interacts with myosin II when MRLC is phosphorylated. Together with these results, this suggests that Drak phosphorylation of Sqh might be necessary for Anillin-mediated myosin II organization within the contractile ring (Chougule, 2016).

Phosphorylation of MRLC on Serine-19 or Serine-19 and Threonine-18 leads to the
unfolding of inactive myosin II hexamers into an open conformation that allows assembly of bipolar myosin II filaments and their association with F-actin to form actomyosin filaments. This is likely how Drak organizes myosin II. Phosphorylation of MRLC on Serine-19 or Serine-19 and Threonine-18 also leads to the activation of the Mg2+-ATPase activity of myosin II that slides actin filaments past each other, causing actomyosin contraction. Three aspects of the drak mutant phenotype support the requirement for Drak in actomyosin contraction: wavy cellularization fronts caused by non-uniform furrow canal depths, abnormal microfilament ring shapes, and failure of microfilament rings to constrict during late cellularization. These are the same defects that suggest an actomyosin contraction defect in src64 mutant embryos. However, src64 mutant embryos do not show myosin II organization defects. Because effective actomyosin contraction likely requires properly organized actomyosin filaments within the contractile ring apparatus, it is unclear whether Drak directly regulates actomyosin contraction or whether Drak only enables actomyosin contraction through proper organization of myosin II within the microfilament rings. One possibility is that phosphorylation of Sqh by Drak both organizes actomyosin filaments into a contractile ring apparatus and directs actomyosin contraction. An alternative possibility is that Drak is directly responsible for organizing actomyosin filaments into a contractile ring by phosphorylating Sqh, but Drak is not directly involved in its contraction and different kinases that phosphorylate Sqh regulate actomyosin contraction. Thus, Drak could be an early regulator of myosin II activity
during cellularization, such that further phosphorylation of Sqh and myosin II-driven contraction is dependent on Drak-mediated organization of myosin II. At some level the regulation of actomyosin contraction diverges from the regulation of actomyosin filament organization: Src64 is required for contraction, but has no role in myosin II organization (Chougule, 2016).

Rescue of myosin II organization, actomyosin contraction and F-actin distribution defects in drak mutant embryos by the mono-phosphorylated SqhE21 phosphomimetic suggests that Drak-mediated mono-phosphorylation of Sqh at Serine-21 is sufficient for regulation of actomyosin dynamics during cellularization. Although the diphosphorylated SqhE20E21 phosphomimetic also rescues myosin II organization and actomyosin contraction defects, it does not rescue F-actin distribution defects in drak mutant embryos. These results are consistent with Drak primarily phosphorylating Sqh at Serine-21, and are consistent with reports that DAPK family members phosphorylate MRLC mainly at Serine-19 (Chougule, 2016).

The normal teardrop shape of the furrow canals in early cellularization is likely caused by actomyosin contraction in the microfilament rings. In drak mutant embryos, unexpanded early cellularization furrow canals and failure of many late cellularization furrow canals to expand further suggest that Drak is required for proper furrow canal structure. Some of the furrow canal structural defects in drak mutant embryos are similar to those of nullo mutant embryos: collapsed furrow canals and blebbing. However, nullo mutant embryos, as well as RhoGEF2 or dia mutant embryos, have other, more severe furrow canal defects: missing or regressing furrow canals and compromised lateral membrane-furrow canal compartment boundaries. Furthermore, cytochalasin treatment causes similar defects, suggesting that reduced F-actin levels in the furrow canals are responsible for these defects. Thus Nullo, RhoGEF2 and Dia regulate F-actin and its levels in furrow canals. These observations suggest that Drak regulates myosin II and thereby regulates actomyosin organization and contraction, and that these are necessary for structural integrity and expansion of the furrow canals, but not for their continued existence (Chougule, 2016).

The furrow canals of drak mutant embryos during late cellularization show extensive blebbing into the lumens. This is consistent with a defect in furrow canal membrane or cortex integrity. Blebs can be formed by local rupture of the cortical cytoskeleton or detachment of the plasma membrane from the cortical actomyosin cytoskeleton. Actomyosin contraction has been implicated in bleb formation. Therefore, it is proposed that blebbing in furrow canals is caused by aberrant localized actomyosin contraction during late cellularization in the disorganized actomyosin cytoskeleton of drak mutant embryos. Contraction is presumably driven by phosphorylation of Sqh by kinases other than Drak. Since actomyosin contraction occurs in a disorganized actomyosin cytoskeleton, it does not lead to uniform constriction of the microfilament rings, but instead leads to localized contraction that produces cytoplasmic blebs. However, other causes for furrow canal defects are possible. Plasma membrane attachment sites might not form or function properly in the disorganized furrow canal cytoskeleton in drak mutant embryos. The disorganized cytoskeleton might inhibit vesicle trafficking. Vesicle trafficking itself might be
defective: mammalian DAPKs have been shown to be involved in membrane trafficking and in phosphorylation of syntaxin A1.
Vesiculated lateral plasma membrane in drak mutant embryos during late cellularization suggests that the plasma membrane breaks down. Intriguingly, scra mutant embryos have lines of vesicles where the closely apposed lateral plasma membranes would have been. However in scra mutant embryos, vesiculation is observed during early cellularization, but to a lesser extent than during late cellularization. drak mutant embryos do not show lateral plasma membrane vesiculation defects until late cellularization. drak mutant defects in both the furrow canal membrane and the lateral plasma membrane might reflect a general defect in membrane integrity. It will be interesting to investigate the potential role of myosin II organization in furrow canal structure and plasma membrane integrity (Chougule, 2016).

Anillin is unequally required during asymmetric cell divisions of the PNS

During Drosophila embryogenesis, timely and orderly asymmetric cell divisions ensure the correct number of each cell type that make up the sensory organs of the larval peripheral nervous system. A role is reported for scraps, Drosophila Anillin, during these divisions. Anillin, a constitutive member of the contractile ring is essential for cytokinesis in Drosophila and vertebrates. During embryogenesis it was found that zygotically transcribed scraps is required specifically for the unequal cell divisions, those in which cytokinesis occurs in an 'off-centred' manner, of the pIIb and pIIIb neuronal precursor cells, but not the equal cell divisions of the lineage related precursor cells. Complementation and genetic rescue studies demonstrate this effect results from zygotic scraps and leads to polyploidy, ectopic mitosis, and loss of the neuronal precursor daughter cells. The net result of which is the formation of incomplete sense organs and embryonic lethality (OFarrell, 2008).

scraps mutation is the source of both the embryonic lethality and PNS phenotype (firmly linking the two phenotypes) associated with the l(2)k08255 chromosome. Zygotic alleles escape the early cellularisation defects via maternally contributed wildtype scraps and instead later suffer severe PNS (and CNS) phenotypes. Notably, neuronal specific expression of scraps was capable of rescuing embryonic lethality. While these larvae likely do not have a completely restored PNS or CNS, the level of restoration achieved is sufficient for survival of the animals to pupal stages. Thus demonstrating that the lack of Anillin within nervous tissues accounted for these phenotypes. High levels of Anillin at critical time-points are likely required in multiple tissues during formation of adult structures, possibly inadequately supplied via the transgenic set-up (OFarrell, 2008).

Anillin is a key component of the actomyosin contractile ring and one of the earliest detectable contractile ring proteins, potentially mediating contact with the cellular membrane and targeting other proteins. As such Anillin represents a candidate for contractile ring nucleation and hence positioning. Anillin has been shown to fulfil such a role in both fission yeast and C. elegans, although no such role has been demonstrated in Drosophila or higher eukaryotes. Thus Anillin is involved in cytokinesis in several systems although the nature of that role may vary. The consequence of lacking zygotic scraps/Anillin during PNS asymmetric cell divisions was examined in this study (OFarrell, 2008).

While some observations were in line with those made in other systems, e.g. the enlargement of cells resulting from failed cytokinesis and the associated multi-nucleate phenotypes, other findings were more surprising. A loss of cells was observed stemming specifically from the pIIb neuronal lineage of the Ch- and ES-organs, while pIIa cell divisions were unaffected. A potential caveat to this observation is the maternal contribution of Anillin, which potentially persists during the PNS divisions. However, since the pIIb divides prior to the pIIa and since the pIIa cell division is unperturbed, this explanation was found to be inadequate to account for the differences observed. A perdurance of maternally contributed protein (or RNA) would be ubiquitous, rather than cell specific. The alternate side of this argument then begs the question; do the SOP and pIIa then not require Anillin? This is also unlikely. Possible explanations to reconcile these two arguments include the differential inheritance of maternally contributed scraps mRNA or protein to the pIIa cell, such mechanisms exist. Alternatively, differential degradation of the Anillin protein within pIIb cells specifically, could account for the differences observed. Finally, as the pIIb cell undergoes unequal cell division and hence contractile ring positioning 'off-center', there could be a requirement for additional, or a specifically modified version of the Anillin protein, supplied via zygotic transcription (OFarrell, 2008).

Interestingly, the resulting polyploid pIIb and pIIIb cells that have failed to divide are capable of differentiating into neurons while simultaneously progressing through the cell cycle. This is likely a re-entry into the cell cycle following a failed cell division event rather than a cell cycle regulatory role for Anillin. Direct links between polyploidy and uncontrolled proliferation have been previously described. A biased requirement for Anillin in unequal pIIb cell divisions is a novel finding, the nature and purpose of which is open to speculation. Further study of the role of Anillin in other models of asymmetric divisions or nervous system development could prove enlightening (OFarrell, 2008).

Anillin is a scaffold protein that links RhoA, actin, and myosin during cytokinesis

Cell division after mitosis is mediated by ingression of an actomyosin-based contractile ring. The active, GTP-bound form of the small GTPase RhoA is a key regulator of contractile-ring formation. RhoA concentrates at the equatorial cell cortex at the site of the nascent cleavage furrow. During cytokinesis, RhoA is activated by its RhoGEF, ECT2. Once activated, RhoA promotes nucleation, elongation, and sliding of actin filaments through the coordinated activation of both formin proteins and myosin II motors. Anillin is a 124 kDa protein that is highly concentrated in the cleavage furrow in numerous animal cells in a pattern that resembles that of RhoA. Although anillin contains conserved N-terminal actin and myosin binding domains and a PH domain at the C terminus, its mechanism of action during cytokinesis remains unclear. This study shows that human anillin contains a conserved C-terminal domain that is essential for its function and localization. This domain shares homology with the RhoA binding protein Rhotekin and directly interacts with RhoA. Further, anillin is required to maintain active myosin in the equatorial plane during cytokinesis, suggesting it functions as a scaffold protein to link RhoA with the ring components actin and myosin. Although furrows can form and initiate ingression in the absence of anillin, furrows cannot form in anillin-depleted cells in which the central spindle is also disrupted, revealing that anillin can also act at an early stage of cytokinesis (Piekny, 2008).

Various organisms might employ distinct mechanisms to recruit anillin, which executes its conserved function as a scaffold protein in the contractile ring. In both S. pombe and animal cells, anillin-like proteins are early markers of the nascent cleavage furrow. In most systems, anillin is not required for furrow formation. C. elegans have three anillin-like proteins, and ani-1 is required for contractile events in the early embryo, such as membrane ruffling and pseudocleavage, but is not strictly required for cytokinesis. Drosophila anillin is required for cellularization in early embryos and promotes late cytokinetic events in S2 cells. Fission yeast express two anillin-related proteins, Mid1p and Mid2p, which regulate proper positioning of the division plane and septation, respectively. Whereas in human cells anillin localizes via its C-terminal Rho binding domain, in S. pombe mid1p localization involves a C-terminal amphipathic helix. Interestingly, the AHD is not well conserved in mid1p, nor is there compelling evidence that RhoA triggers contractile-ring formation in S. pombe (Piekny, 2008).

Anillin-mediated targeting of Peanut to pseudocleavage furrows is regulated by the GTPase Ran

During early development in Drosophila, pseudocleavage furrows in the syncytial embryo prevent contact between neighboring spindles, thereby ensuring proper chromosome segregation. This study demonstrates that the GTPase Ran regulates pseudocleavage furrow organization. Ran can exert control on pseudocleavage furrows independently of its role in regulating the microtubule cytoskeleton. Disruption of the Ran pathway prevents pseudocleavage furrow formation and restricted the depth and duration of furrow ingression of those pseudocleavage furrows that form. Ran is required for the localization of the septin Peanut to the pseudocleavage furrow, but not anillin or actin. Biochemical assays revealed that the direct binding of the nuclear transport receptors importin α and importin β to anillin prevents the binding of Peanut to anillin. Furthermore, RanGTP reverses the inhibitory action of importin α and β. On expression of a mutant form of anillin that lacks an importin α and β binding site, inhibition of Ran no longer restricts the depth and duration of furrow ingression in those pseudocleavage furrows that form. These data suggest that anillin and Peanut are involved in pseudocleavage furrow ingression in syncytial embryos and that this process is regulated by Ran (Silverman-Gavrila, 2008).

During cytokinesis, the ingressing plasma membrane physically divides the mother cell into two daughter cells. Membrane ingression during cell division is both temporally and spatially regulated, ensuring that membrane scission occurs (1) only after the chromosomes have fully segregated and (2) between the two chromosomal masses. The signals within the cell that determine cytokinetic furrow positioning are complex, reflecting the strict control needed to ensure that cytokinesis is successful. Signals from astral microtubules, the spindle midbody, the nucleus, and the membrane itself direct the assembly of the contractile ring to the equatorial cortex of the plasma membrane. The contractile ring is an actomyosin-based structure that constricts and generates the force needed to drive membrane ingression. As the membrane ingresses, it is remodeled and stabilized (Silverman-Gavrila, 2008).

Other membrane ingression events share many of the same features and involve many of the same proteins as cytokinetic furrows. In the syncytial Drosophila embryo before cellularization, up to 6000 closely packed nuclei exist in a common cytosol close to the cortex. To ensure faithful chromosome segregation during the rapid nuclear divisions, nuclei are isolated from one another to prevent neighboring spindles from contacting and fusing. To achieve this, plasma membrane ingressions form transiently between nuclei during the rapid nuclear cycles before cellularization. These membrane ingressions, termed pseudocleavage or metaphase furrows, are organized by the actin cytoskeleton and bear a close resemblance to cytokinetic cleavage furrows. First actin caps form at the plasma membrane above each nucleus. Then during interphase, as the centrosomes migrate to either side of the nucleus, the actin caps expand correspondingly. In prophase the cap reorganizes to drive membrane ingression into the embryo such that nuclei and newly forming spindles are separated from one another. Toward the end of metaphase, the furrows begin to retract and dissipate by anaphase. This process is repeated from the tenth through the thirteenth nuclear cycles. During the fourteenth nuclear cycle, the syncytial embryo cellularizes to form 6000 columnar epithelial cells. In this instance the cleavage furrows extend down into the embryo, before growing transversally and fusing to form a single layer of nucleated cells (Silverman-Gavrila, 2008).

Most components required for furrow ingression are conserved between cytokinetic furrows (during conventional mitosis) and pseudocleavage furrows. However, there are some differences. Notably pseudocleavage furrows are membrane ingressions that do not meet and therefore do not lead to membrane fusion. Instead they extend into the embryo, perpendicular to the cortex, and then retract back toward the embryo cortex after the chromosomes have begun to segregate. In addition, there is a difference in the stage of the cell cycle when the furrow components assemble. Although the cytokinetic furrow begins to assemble during anaphase and is required to divide a cell in two, the syncytial embryo pseudocleavage furrows begin to assemble in prophase and serve to prevent neighboring spindles from contacting one another (Silverman-Gavrila, 2008).

A key protein involved in cytokinetic furrow function is anillin, which has multiple domains allowing it to bind and bundle actin filaments, target septins to the plasma membrane, and interact with components of the microtubule-bound centralspindilin complex. Consequently anillin is thought to act as a scaffold for the correct assembly of the contractile ring. It is not fully understood how the role of anillin in cytokinesis is regulated. However, its role in remodeling the actomyosin contractile ring in somatic cells is in part regulated by its differential spatial positioning in the cell during the cell cycle. In interphase anillin localizes to the nucleus where it cannot interact with actin and myosin at the plasma membrane. However, in mitosis upon nuclear envelope breakdown, anillin is released from the nucleus and is targeted to the cortex of the plasma membrane and later to the equatorial cortex of the plasma membrane in a RhoGTP-dependent manner. The spatial regulation of anillin during the cell cycle contributes to the restriction of its function to mitosis. However, in Drosophila syncytial embryos anillin is cytosolic, localizing to pseudocleavage furrows throughout the nuclear cycle, suggesting that it may be regulated by other mechanisms (Silverman-Gavrila, 2008).

One function of anillin is to target septins to the contractile ring. Septins are a family of GTP-binding proteins that can assemble into filaments. Septins have been attributed multiple roles: as membrane diffusion barriers, as stabilizers of the furrow, in membrane trafficking, and as a scaffold. In Drosophila there are five septins: Peanut, Sep1, Sep2, Sep4, and Sep5. Peanut, Sep1 and Sep2 have been isolated as a stoichiometric complex that in vitro can polymerize into filaments. In contrast, Xenopus laevis Sept2 can self assemble into filaments, suggesting that septins may function independently (Silverman-Gavrila, 2008 and references therein).

The GTPase Ran is a key positive regulator of mitosis (Ciciarello, 2007). RanGTP regulates a number of mitotic factors that are sequestered in the nucleus by nuclear transport receptors during interphase. In mitosis RanGTP antagonizes the binding of nuclear transport receptors to these proteins and thereby promotes their activity. RanGTP is at its highest concentration around the chromosomes, where RCC1 the nucleotide exchange factor for Ran is localized. Consequently, RanGTP has been proposed to act as a spatial cue by only activating these mitotic proteins close to the chromosomes (Caudron, 2005; Kalab, 2006). In so doing RanGTP is thought to specify where certain mitotic processes occur in the cell. For example, it could specify that spindle assembly only occurs around chromosomes. The full extent to which this mechanism regulates the mitotic cell is not known and continues to expand (Silverman-Gavrila, 2008).

This study demonstrates a new role for Ran in regulating pseudocleavage furrow ingression, a membrane invagination process in early Drosophila embryos. The Ran pathway regulates the interaction between anillin and the septin Peanut, thereby regulating furrow stability (Silverman-Gavrila, 2008).

A cytological screen was carried out to identify mitotic processes regulated by the Ran pathway. Inhibitors of the Ran pathway were injected into GFP-α-tubulin-expressing embryos just before mitotic entry, and then microtubule organization was monitored by time-lapse microscopy. One phenotype, the fusion of neighboring spindles, occurred more frequently upon the injection of inhibitors of the Ran pathway compared with control injections. In control injected embryos 0.2% of observed spindles fused to a neighboring spindle. In contrast, inhibition of the Ran pathway by injecting either the dominant negative allele of Ran, RanT24N, or importin α resulted in 8.4 and 7.8% of observed spindles fusing to neighboring spindles, respectively (Silverman-Gavrila, 2008).

Peanut is recruited to ingressing furrows by anillin, a multifunctional protein required for cytokinesis that interacts with myosin II, actin, and septins. Septins bind to the carboxy-terminus of anillin, which includes a pleckstrin homology (PH) domain. Drosophila anillin has three potential nuclear localization signals (NLS) that could bind to the nuclear transport receptors importin α and β. Two of the NLS motifs are located in or directly adjacent to the PH domain (Silverman-Gavrila, 2008).

To determine if the carboxy-terminus of anillin could bind to importin α and β, a fusion was constructed between GST and the carboxy-terminus of anillin (amino acids 815-1201, anillin-CT, and its ability to bind to recombinant importin α and β was analyzed. Both importin α and β bound to anillin-CT, and this binding was reversed in the presence of RanQ69L, a point mutant of Ran locked in the GTP-bound state. Of the two potential NLS motifs, the one located between amino acid residues 989 and 999, bares the closest resemblance to an archetypal bipartite NLS and is found in the same region of human anillin (amino acids 887-898). Mutation of lysines 997-999 to alanine (3A-anillin-CT) abrogate both importin α and β binding to this region of anillin, suggesting that amino acids 989-999 constitute a nuclear transport receptor-binding site (Silverman-Gavrila, 2008).

It was next asked if the anillin-CT could interact with Peanut. GST-anillin-CT was incubated with 0-3-h Drosophila embryo extract and then isolated using glutathione agarose beads. Anillin-CT copurified with Peanut and another septin, Sep2. However, the addition of exogenous importin α and importin β inhibited the binding of Peanut to anillin-CT in a concentration- and NLS-dependent manner. This inhibition was specific to Peanut, because Sep2 binding to anillin-CT was not inhibited by importins (Silverman-Gavrila, 2008).

To determine if the in vivo targeting of Peanut and Sep2 to the pseudocleavage furrows is differentially regulated, importin α was injected into syncytial embryos and GFP-Sep2 localization was determined by time-lapse microscopy. Consistent with in vitro results, GFP-Sep2 localization was not perturbed upon interfering with the Ran pathway. Furthermore, in fixed GFP-Sep2-expressing embryos in which the Ran pathway has been perturbed, Peanut fails to localize to nascent furrows, whereas GFP-Sep2 does localize to nascent furrows. These data suggest that Peanut and Sep2 are differentially regulated by Ran and that Sep2 can localize to pseudocleavage furrows independently of Peanut (Silverman-Gavrila, 2008).

This study has identified RanGTP as a regulator of the interaction between Peanut and anillin. This mechanism operates directly and independently of Ran's well-characterized role in regulating the mitotic microtubule cytoskeleton (Silverman-Gavrila, 2008).

Studies suggest that anillin is required for the recruitment of septins to the furrow. By perturbing the Ran pathway, this study has demonstrated that the recruitment of the septins Peanut and Sep2 is differentially regulated, consistent with previous observations that Sep1 recruitment to furrows is dependent on Peanut but Sep2 is not. Anillin lacking the importin binding site between residues 997 and 999 can bind to Peanut in the presence of importins, suggesting that importins directly block the anillin-Peanut interaction rather than disrupting the Peanut, Sep1, and Sep2 complex. These data suggest that although Peanut, Sep1, and Sep2 can exist in a single complex, they may be able to function independently of one another as has been demonstrated in vitro for a Xenopus septin (Silverman-Gavrila, 2008).

Perturbing the Ran pathway destabilizes pseudocleavage furrows. One mechanism for this is through the regulation of the anillin-Peanut interaction. In embryos that expressed an anillin mutant lacking the importin-binding site, Peanut recruitment to pseudocleavage furrows occurs even in the presence of exogenous importins, and furrows demonstrate wild-type dynamics. These data suggest that Peanut is required for pseudocleavage furrow stability. This role for anillin and Peanut is consistent with the observed role for these proteins in stabilizing the cellularization furrow later in Drosophila development. These findings may at first appear to contradict those studies, in which embryos lacking Peanut protein progressed through the syncytial nuclear divisions only showing the first defects during cellularization. However, these studies only analyzed syncytial furrows from the top, apical view and not from the lateral view to observe ingression dynamics. Therefore, these studies would not have detected changes in furrow ingression dynamics that were observed upon inhibition of Ran, which correlated with a failure to recruit Peanut to the furrow (Silverman-Gavrila, 2008).

The Ran pathway regulates pseudocleavage furrow ingression directly by regulating importin binding to anillin. It was previously shown that in Drosophila syncytial embryos the importin β, whose injection causes similar effects as importin α, is released from the nucleus upon nuclear envelope breakdown and becomes diffuse throughout the cytosol during the rest of mitosis. During this period pseudocleavage furrows begin to retract. Therefore, as importin β is cytosolic during metaphase and anaphase it could act to prevent the interaction of Peanut and anillin. In turn this would lead to furrow instability and retraction (Silverman-Gavrila, 2008).

It cannot unequivocally be ruled out that some of the defects caused by perturbing the Ran pathway are due to a disruption of microtubule cytoskeleton. Indeed, one microtubule-dependent furrow phenotype, the formation of pseudocleavage furrows that encompassed a small area of cytosol around a nucleus, was observed. This phenotype has also seen in another study upon depolymerization of microtubules in embryos. However, microtubule depolymerization when instigated in interphase does not cause a failure in pseudocleavage furrow formation, a finding consistent with a previous study (Silverman-Gavrila, 2008).

Another mechanism through which Ran could affect pseudocleavage furrows is by disrupting nuclear trafficking. Indeed it was observed that nuclear trafficking can be reduced by up to 50% upon disruption of the Ran pathway. However, it seems unlikely that the changes in nuclear import kinetics in these experiments disrupted the function of anillin because anillin is a cytosolic protein in the syncytial embryo and localizes to the leading edge of the ingressing furrow during interphase. It is not understood how anillin is retained in the cytoplasm of syncytial embryos because it is imported into nuclei in other developmental stages. However, this phenomenon is not unique to anillin and is also exhibited by the kinesin Pavarotti, another protein involved in pseudocleavage furrow organization (Silverman-Gavrila, 2008).

These studies suggest that Ran regulates multiple factors involved in pseudocleavage furrow ingression, because embryos expressing the mutant anillin still exhibit a failure to form all the expected pseudocleavage furrows. Failure to fully suppress the phenotype could be due to the continued presence of endogenous anillin or reflect that other Ran pathway-sensitive factors are involved in pseudocleavage furrow formation. Regulation through the Ran pathway could define a spatial cue concentrated around chromosomes and extending to the cortex. Such a spatiotemporal regulatory mechanism could be involved in promoting cytokinetic furrows in other cells. A recent study in oocytes finds that Ran regulates myosin II, whose activity is required for cytokinetic cleavage furrows. In addition importin α is required for ring canal organization during oogenesis. Ring canals form as a result of incomplete cytokinesis, and many proteins involved in cytokinesis both localize to and are required for their formation, including anillin and septins (Silverman-Gavrila, 2008).

The data suggest that the anillin-Peanut interaction, which is inhibited by importins must occur in regions of the cell where there are low levels of importins or high levels of RanGTP. Recent studies have visualized a RanGTP-importin β gradient and found that it persists from the chromosomes to the centrosomes, a distance similar to that between the metaphase plate and the cortex. Thus, RanGTP could play an important role in positioning the plane of cleavage by defining on the cell cortex where furrow proteins interact (Silverman-Gavrila, 2008).

Although there are clear differences between cytokinetic and pseudocleavage furrows, anillin and septins are involved in both. Therefore, this study suggests that Ran could also have a role in regulating cytokinetic furrows. Whether chromosomes play a significant role in cytokinesis remains controversial. However, studies where nuclei or chromosomes are asymmetrically positioned within a cell show that furrow ingression coincided with the region of the cell that contained the chromosomes, suggesting that signals from the nucleus and in particular the chromosomes had a role in specifying furrow ingression. Similarly, enucleated sea urchin eggs are able to duplicate their centrosomes and generate astral arrays of microtubules, but fail to form stable cleavage furrows. The current study proposes a molecular mechanism to explain, at least in part, these observations, suggesting that RanGTP generated around the chromosomes is a diffusible signal that facilitates multiple processes required for furrow formation. Whether RanGTP is required early in cytokinesis to 'prime' the cortex for a future ingression or acts directly later during the ingression process is unclear. Testing these hypotheses is not straightforward, since Ran is also required for organizing the mitotic microtubule cytoskeleton, which is required for cytokinesis. Taken together these findings suggest an additional mechanism involved in regulating cytokinesis that is dependent on signals from chromosomes in addition to those stemming from the different organizational states of the mitotic microtubule cytoskeleton (Silverman-Gavrila, 2008).

Spatial control of cytokinesis by Cdr2 kinase and Mid1/anillin nuclear export

Maintaining genome integrity and cellular function requires proper positioning of the cell division plane. In most eukaryotes, cytokinesis relies on a contractile actomyosin ring positioned by intrinsic spatial signals that are poorly defined at the molecular level. Fission yeast cells assemble a medial contractile ring in response to positive spatial cues from the nucleus at the cell center and negative spatial cues from the cell tips. These signals control the localization of the anillin-like protein Mid1, which defines the position of the division plane at the medial cortex, where it recruits contractile-ring components at mitosis onset. This study shows that Cdr2 kinase anchors Mid1 at the medial cortex during interphase through association with the Mid1 N terminus. This association underlies the negative regulation of Mid1 distribution by cell tips. The positive signaling from the nucleus is based on Mid1 nuclear export, which links division-plane position to nuclear position during early mitosis. After nuclear displacement, Mid1 nuclear export is dominant over Cdr2-dependent positioning of Mid1. It is concluded that Cdr2- and nuclear export-dependent positioning of Mid1 constitute two overlapping mechanisms that relay cell polarity and nuclear positional information to ensure proper division-plane specification (Almonacid, 2009).

This study shows that Mid1 delivery to the central cortex depends on two overlapping mechanisms that ensure specification of the division plane in the middle of the cell. These two mechanisms relay distinct positional information on cell polarity and nuclear localization and lead to equal partitioning of the cytoplasm and correct segregation of chromosomes. When the two mechanisms are spatially separated by nuclear displacement, Mid1 nuclear export, which operates later during the cell cycle, is dominant over Cdr2-dependent positioning of Mid1. Overlapping regulatory systems appear to underlie spatial regulation of cytokinesis in a range of cell types. For example, the Caenorhabditis elegans cell division plane is positioned by consecutive spatial signals from astral microtubules and the spindle midzone. One target of these signals is anillin, which serves as a scaffold to build the contractile ring and is functionally related to Mid1. Such overlapping systems provide robust spatial control of cytokinesis to maintain genomic stability (Almonacid, 2009).

In eukaryotes, cytokinesis generally involves an actomyosin ring, the contraction of which promotes daughter cell segregation. Assembly of the contractile ring is tightly controlled in space and time. In the fission yeast, contractile ring components are first organized by the anillin-like protein Mid1 into medial cortical nodes. These nodes then coalesce laterally into a functional contractile ring. Although Mid1 is present at the medial cortex throughout G2, recruitment of contractile ring components to nodes starts only at mitotic onset, indicating that this event is cell-cycle regulated. Polo kinases are key temporal coordinators of mitosis and cytokinesis, and the Polo-like kinase Plo1 is known to activate Mid1 nuclear export at mitotic onset, coupling division plane specification to nuclear positio. This study provide evidence that yeasts Plo1 also triggers the recruitment of contractile ring components into medial cortical nodes. Plo1 binds at least two independent sites on Mid1, including a consensus site phosphorylated by Cdc2. Plo1 phosphorylates several residues within the first 100 amino acids of Mid1, which directly interact with the IQGAP Rng2, and influences the timing of myosin II recruitment. Plo1 thereby facilitates contractile ring assembly at mitotic onset (Almonacid, 2011).

Intercellular protein movement in syncytial Drosophila follicle cells

Ring canals connecting Drosophila germline, follicle and imaginal disc cells provide direct contact of cytoplasm between cells. To date, little is known about the formation, structure, or function of the somatic ring canals present in follicle and imaginal disc cells. This study shows by confocal and electron microscopy that Pavarotti kinesin-like protein and Visgun are stable components of somatic ring canals. Using live-cell confocal microscopy, it was shown that somatic ring canals form from the stabilization of mitotic cleavage furrows. In contrast to germline cells, syncytial follicle cells do not divide synchronously, are not maximally branched and their ring canals do not increase in size during egg chamber development. Somatic ring canals permit exchange of cytoplasmic proteins between follicle cells. These results provide insight into the composition and function of ring canals in somatic cells, implying a broader functional significance for syncytial organization of cells outside the germline (Airoldi, 2011).

The persistence of cleavage furrow proteins in ring canals is a common feature of both germline and somatic ring canals. Many of the follicle cell ring-canal components identified to date are proteins with known roles in cytokinesis. Anillin and Pav-KLP accumulate in cleavage furrows as they begin to constrict, and
remain associated with ring canals, as was observed in images of
live dividing follicle cells (Airoldi, 2011).

Mutations in scraps (the gene encoding Anillin) and pav cause
defects in cytokinesis and result in multinucleate cells. In addition, an Anillin-binding protein called Cindr was recently identified as another
component of both cleavage furrows and somatic ring canals (Airoldi, 2011).

The data helps refine the list of somatic ring canal
proteins; Vsg can be added, and Nasrat and Polehole can be eliminate.
GFP::Vsg was reported to be present in germline ring canals and
follicle cell puncta. The localization of GFP::Vsg to follicle cell
ring canals was confirmed using immunoEM and colocalization studies. Vsg is a
predicted sialomucin protein with a single transmembrane
domain and multiple predicted extracellular O-linked
glycosylation sites. Its localization to somatic ring canals is
highly reminiscent of Mucin-D localization; however, it was not possible to confirm this directly because Mucin-D antibodies are no longer available. Vsg
(and Mucin-D) are also present in ring canals of larval imaginal
disc and brains. Vsg protein bears predicted localization signals,
including an N-terminal plasma membrane localization signal
and a C-terminal vesicular trafficking sequence. Therefore, its
specific localization to ring canals could indicate a role of vesicular trafficking in the formation of stable intercellular bridges (Airoldi, 2011).

Previous work on Vsg revealed roles in promoting cell
proliferation and embryonic development, though its specific
function is unclear. Its vertebrate homolog, endolyn, is targeted to endosomes and lysosomes, and functions in the maintenance of hematopoietic progenitors and myoblast fusion. The highest degree of similarity between Vsg and Endolyn is in the transmembrane and cytoplasmic domains, including the C-terminal lysosomal
sorting signals, suggesting a common underlying role for this
family of sialomucins (Airoldi, 2011).

Not all follicle cells have stable ring canals
The data indicate that 70% of follicle cell divisions within the
germarium result in a stable ring canal, and this increases to 89%
of divisions outside the germarium. A possible explanation for
the absence of ring canals is that a percentage of cell divisions
complete cytokinesis and a ring canal is never formed. In light of
the data, this would suggest that follicle cells within the
germarium are more likely to complete abscission, possibly
because these cells are still in the early stages of differentiation
and still contain factors that promote normal cell division (Airoldi, 2011).

Alternatively, the absence of ring canals that wsd observed might
be a result of instability or destruction of otherwise normal ring
canals, or to the merging of existing ring canals. If so, the higher rate of ring canal loss in the germarium could be attributed to the mechanical stresses of migration as the follicle cells move to encapsulate the germline cyst and
reposition for stalk formation. Once outside the germarium, the
constant but smaller rate of ring canal loss could be a result of shuffling within the epithelium, cell death or eventual resolution of cytokinesis (Airoldi, 2011).

Specialized subpopulations of follicle cells lack ring canals.
No ring canals were observed in mature stalk cells; however,
ring canals were observed between cells in the region of the
forming stalk as egg chambers exit the germarium.
Loss of ring canals during stalk formation could be due to
mechanical disruption as cells intercalate to form a single chain
of cells. A model of mechanical disruption of existing ring canals
is further supported by observations of border cells.
Presumptive border cells in stage 8 egg chambers have normal
ring canals, but migrating border cells contain fewer, smaller, and
apparently fragmented GFP::Pav-KLP puncta (Airoldi, 2011).

No ring canals were observed in pairs of polar cells. This is
not surprising, because the polar cell precursors are specified
while in the germarium and do not continue to divide. Furthermore, the precursor population is reduced to only two cells through programmed cell death
between the germarium and stage 5,
which would further isolate the polar cells. However, additional
investigation is necessary to determine whether the polar cell
precursors have ring canals, either between themselves or with
other main-body follicle cells (Airoldi, 2011).

Given the presence of persistent intercellular bridges within
follicle cell lineages, whether follicle cell mitotic
divisions are synchronized, as they are in male and female
germline cells, was investigated. These results clearly indicate that some level of
synchrony exists between small groups of follicle cells because
adjacent mitotic cells are significantly more frequent than
predicted from a random distribution. The coordination of mitosis could be carried out by a cell nonautonomous mechanism in which mitosis-promoting factors pass
through ring canals and initiate mitosis in the next cell. However,
nearly all mitotic cells had ring canals connecting them to nonmitotic
cells, so any propagation of mitosis-promoting signals
would be limited. Furthermore, mitotic divisions within follicle
cell lineages consisting of hundreds of cells are far from fully
synchronized, suggesting that any influence of neighbors on entry
into mitosis is weak. Alternatively, a cell-autonomous cell cycle
'timer' could be responsible for the apparent coordination of
mitosis between sibling cells, regardless of a connecting ring
canal. In this scenario, two sibling cells would enter mitosis
together because their cell cycles were synchronized at the
conclusion of the previous division. Of note, it was found that 47%
of cells were in mitosis independently of all neighboring cells,
which implies that if a cell-autonomous cell cycle timer controls
entry into mitosis, the length of the overall cell cycle (9.6 hours)
will vary by more than 24 minutes in nearly half of all cells. The
data are insufficient to distinguish between cytoplasm sharing
and timer mechanisms, because the existence of ring canals
between neighboring mitotic cells fits both models (Airoldi, 2011).

The pattern of connections between cells with ring canals is
determined by spindle orientation with respect to ring canals
from previous mitoses. In Drosophila germline mitoses, spindle
orientation and thus ring canal inheritance is controlled by the
fusome, a cytoplasmic organelle containing ER membranes that
extends through each intercellular bridge. After four mitotic divisions, the 16 cells are maximally branched with the original two cells having four ring
canals each and the youngest eight cells from the final division
each having one ring canal. By contrast, the follicle cells, which
do not have a fusome, appear to divide randomly with respect to
pre-existing ring canals, and their pattern of connections is
intermediate between fully branched and completely linear. Other
mechanisms are likely to contribute to the pattern of follicle cell
divisions, such as a limitation on how many direct connections an
individual cell can have to hexagonally packed neighbors (Airoldi, 2011).

The presence in follicle cells of intercellular bridges large enough
for the passage of small vesicles presents the possibility of
extensive flow of information between these cells. However,
many aspects of follicle cell biology strongly suggest a cell-autonomous
system. For example, small subsets of follicle cells
pattern the oocyte during development, an essential function that would be
compromised by unrestricted spreading of localized signals to
neighboring cells. Also, genetically mosaic patches of follicle
cells appear to be reliably marked by expression of various
reporters. Follicle cells are also known to have highly mosaic
expression of many proteins, some of which clearly maintain
very different levels of expression despite a connecting ring canal (Airoldi, 2011).

As a result, it was surprising to discover evidence for
robust and rapid exchange of PA-GFP between cells, in patterns
consistent with syncytia of follicle cells connected by ring canals.
Importantly, this result was confirmed by observing movement of
tagged endogenous proteins by FLIP. In both sets of experiments
with fluorescent proteins, exchange of protein was observed in one
or two immediate neighbors of the target cell. Not only does this
argue against nonspecific activation or bleaching, it is also
consistent with the number of ring canals per cell that were
observed in other experiments. Co-expression of GFP::Oda and
GFP::Pav-KLP confirmed these results by demonstrating that
protein movement occurs only between cells connected by a ring canal (Airoldi, 2011).

Of note, FLIP data also show that some proteins are not
exchanged through ring canals, whereas others pass freely.
Components of large protein complexes, such as ribosomal
proteins and those involved in mRNA biogenesis, appear
restricted in their movement. By contrast, orinithine
decarboxylase antizyme and calmodulin are relatively small
(40-60 kDa), monomeric proteins, and exchange between cells
rapidly. This suggests that large and/or highly complexed
proteins do not exchange between cells at a high rate, but smaller proteins do. These results point to the possibility of extensive intercellular movement of protein among somatic cells connected by ring canals (Airoldi, 2011).

Emerging data now reveal that stable ring canals extensively
interconnect follicle cell lineages. In addition, similar ring canals
connect cells of the imaginal discs and larval brain. These
somatic ring canals are over 100 times larger than gap junctions
(250 nm for ring canals versus 1.5 nm for gap junctions) and thus
provide a viable path for macromolecules to move between cells.
Live-imaging data show that some proteins can freely
exchange between connected follicle cells, which raises
interesting questions about the true autonomy of these cells.
A practical consideration concerns the use of GFP as a marker
for genetic mosaic analysis, because unrestricted movement of
GFP between cells would compromise it as a clonal marker (Airoldi, 2011).

For example, using GFP loss to identify a mitotic clone of
homozygous mutant cells could be confounded by GFP
movement from a twin-spot cell or an unrecombined
heterozygous cell. However, the data suggest that for large
clones, the movement of GFP is unlikely to affect the results. The
majority (66%) of follicle cells have ring canals to only one or
two other cells, which suggests that the number of potentially
compromised cells would be small. Furthermore, GFP moving
into a genetically mutant cell could be accompanied by wild-type
gene product, potentially creating a wild-type phenotype.
However, if small clones or single cells are being investigated, protein exchange through ring canals might pose a significant concern (Airoldi, 2011).

Further investigation to determine the extent and control of traffic through ring canals will reveal how follicle cells use and regulate their ability to directly communicate with neighboring cells (Airoldi, 2011).

Paoletti, A. and Chang, F. (2000). Analysis of mid1p, a protein required for placement of the cell dividion site reveals a link between the nucleus and the cell surface in fission yeast. Mol. Biol. Cell 8: 2751-2773. PubMed citation: 10930468